Critical error dea1 l9

critical error dea1 l9

of broad OHM components, and represent the most serious source of systematic error in estimating magnetic fields. The best fit fields to these ripples in. Find helpful customer reviews and review ratings for EGate L9 Android HD even most 1080 MP4s and gave unsupported format error, which reminded me of the. If a severe error is detected, the system can terminate CICS even if you specify that CICS is not to be terminated. critical error dea1 l9

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Reviewed in India on 16 January 2022

Style name: L9 Pro-Max FHDVerified Purchase

This is my second Egate Projector (I had Egate I9 Pro before this). I bought it at INR 22k after doing a lot of research and comparing many brands. My requirements are a projector that we can watch even during the day because we do not own a TV.
1. Easy Setup - There is nothing to setup to be honest as this is not an Android projector. The only thing to be setup was the Keystone correction, which is pretty easy with the remote
2. Excellent brightness - It has the best brightness of any projector I have used so far. So much so that we could watch it during daytime with curtains drawn, with as much light in the room that you could eat or the kids can play
3. Picture Quality - It could have been better but it is OK, quite comparable to the cheaper models like i9 pro
4. Digital Keystone correction - It works well, something it's predecessors missed
1. Bulky - It is big in size, i.e. not quite portable as it will not fit in your usual laptop bag
2. Interface - It's boring, they haven't upgraded much since I bought the i9 pro in 2017. Other brand projectors such as Wanbo have a better interface which looks more professional
3. Keystone Correction - The physical keystone correction has the same problems that its predecessors like i9 had, the corners get blurred if you use physical correction
4. Sound - Could have been better. It's better than the cheaper eGate projectors but not enough to watch a movie in hall, it's only sufficient for a small room
5. USB Play - It did not play a lot of formats, even most 1080 MP4s and gave unsupported format error, which reminded me of the irritating same problem with the i9. I expected better from L9
To be honest, I expected more from a projector costing 22k. The only marginal advantage I got was the brightness so that I can watch it during daytime. The worse part is it will always need a physical input because it is not Android and doesn't support Miracast/screen-share, not even a damn bluetooth so you need another output for sound; so hanging this from the ceiling is another pain. I bought i9 for INR5500 and it gave me 1080 picture, critical error dea1 l9, with the drawback that it needed complete darkness for good experience, however as a projector it did what it should for that amount; though it had a shoddy build quality and its polarization filter got burnt in an area within an year, and Egate customer care did not respond in time, by then my warranty had run out. I ordered a Wanbo X1 pro (INR12k) before this and it had better interface, with Miracast and Bluetooth so using it was a critical error dea1 l9 compared to this.
So, buy this only if you have a requirement of using it in daytime, if you plan on watching few movies etc in a dark room, buy a cheaper one because that will do the job.

,    Pages 35-40

, University of Iowa

(student), University of Iowa

Procedures for application of additive conjoint measurement to group rather than individual data are discussed. A statistical interpretation of the model is proposed and a goodness-of-fit test examined.


Additive conjoint measurement has been applied primarily to individual level data in marketing research studies. For example, Green and Rao (1971) and Green and Wind (1975) give many micro-level applications and Johnson (1974) states that since his approach "is concerned with value systems of individual consumers, the method is most appropriate for product categories where consumer desires are heterogeneous and where markets are highly segmented." These individual level applications often result in important insights but if the model is to develop its full potential for practical marketing problems then changes should be made which also allow its critical error dea1 l9 on aggregate data. The purpose of this paper is to discuss several issues in aggregating responses for additive conjoint measurement. These are:

1. An aggregation procedure should retain information about the variation of subjects around a "group" rank.

2. The model presently assumes the ranking judgments are infallible (errorless) rather than fallible.

3. A parametric goodness-of-fit test is needed.

The methods proposed for dealing with these issues are illustrated using the results of a pilot study of product attributes and consumer environmental concern.

Problem Background

The technique currently employed for aggregation is to average the rankings of several individuals and submit this average ranking to a conjoint scaling algorithm, such as MONANOVA (1965). The problem with this technique is its lack of an explicit model relating the individual rankings to the aggregate. Two aspects of such a model would be whether the individual rankings are compatible with a single overall ordering and how to deal with variation in the individual ranks relative to the overall.

A serious issue here as in any model building paradigm is how to test the goodness-of-fit of the model. Using the average rank procedure the goodness-of-fit measure is the same as with a single person; i.e., stress. However the averaging process results in a considerable loss of information about the variation of individuals around an average rank position. Stress disregards this variation and hence is better suited as a goodness-of-fit index when just one ranking is analyzed.

These problems could be solved more reasonably if the additive conjoint model were statistical in nature rather than mathematical. A statistical model would allow for error in the input judgments and yield an explicit parametric goodness-of-fit test. The parametric assumptions required for a statistical model would be somewhat restrictive relative to the nonparametric version as it is presently known. However this added restriction is compensated by gains in the analyst's ability critical error dea1 l9 diagnose when (end why) the model is probably false.


The conjoint measurement model is usually interpreted as deterministic. The choice alternatives are assumed to be composed of m factors with ni levels for factor i. In a complete factorial design a subject would rank order the EQUATION possible "brands" which can be generated by varying over all levels on all factors. The final ranking is assumed to be error free or "infallible."

Thurstone's judgment scaling model provides a basis for conceptualizing "fallibility" in ordinal judgments [Torgerson (1958) Ch. 8]. The statistical assumptions introduced by Thurstone can be interpreted to account for error at two levels: (1) the fallibility of an individual's choice on a single trial relative to some "true" choice over many similar trials and (2) the fallibility of an individual's judgment relative to some "true" judgment of a group.

Individual Fallibility

For case (1) assume an individual must decide which of two alternatives xi critical error dea1 l9 or xj is preferred. [Only the ordinal judgment in a paired-comparison need be discussed since any rank order can be decomposed into a series of binary choices. The relation "is at least as preferred" is denoted >.The tilde, e.g., y, denotes a random variable.] Thurstone assumed the judgment for this pair is not based on the subject's true perception of each alternative but on discriminal signals which are a function of the true perceptions and random error. Denoting the error associated with xi as di, the ordinal judgment is based on the signals

yi = xi + di   and yj = xj + di    (1)

then xi > xj if and only if yi > yj.

The di represent error which might be present due to momentary lapses in concentration, effects of experimental context, physiological distortions and other influences on a particular trial. This is essentially a regression model with each di assumed normally distributed with mean zero and variance si2. These assumptions along with simplifying conditions (e.g., si2 = s2 all i) lead Thurstone to the law of comparative judgment. [Details can be found in (Torgerson, Ch. 8, 9, 10).]

Group Fallibility

The second interpretation of error (in which the analyst aggregates judgments over individuals) is more pertinent for the issues discussed here. The group is assumed to have a true perception for each alternative but individuals in the group may have perceptions which vary around the true one. The error term accounts for this type of fallibility.

In either case the experimental data which summarizes the ordinal judgments is the proportion of times xi > xj. These proportions can be generated by replicating over trials for case 1, over people for case 2 or in combination. Since the emphasis in this paper is to illustrate aggregation over people the second mode is used. An advantage of using paired-comparison proportions versus average ranks in forming a single scale for conjoint measurement is that the resulting ordering can be tested for unidimensionality. [see Torgerson (1958, p. 185)]


Using the judgment scaling assumptions in the context of additive conjoint measurement requires certain interpretations which should be made explicit. Suppose the alternatives to be ranked are composed of just two factors a, b with levels aj (j = 1. . ., r) and bk (k=1. . ., c). Crossing these factors gives the alternatives xi (i = 1. . ., r . c). In the above discussion the error assumptions were placed on the combinations, xi. However the additive conjoint model assumes there exist scale values or utilities for the factor levels which are additive. Denoting these values with "^" the (errorless) model is:

xi = aj + bk   (2)

Since the statistical model assumes the xi are subject to error it is reasonable to relate this to error on the right hand side of equation 2. Several interpretations exist but perhaps the most transparent is to assume possible error in the implicit judgments on each factor. This model is written:

xi + di = (aj  + eaj) + (bk + ebk )   (3)

where; eaj, critical error dea1 l9, ebk  are random error terms associated with levels j and k on a and b respectively.

It is not the purpose here to develop the many consequences of this formulation. Some work has been attempted in this vein by Falmagne (1976). However it is important for the goodness-of-fit test used later to examine some ways the errors on the right hand side of eq. 3 may combine. These are simply listed for reference.

1. The additive independence axiom in an additive conjoint structure (see footnote 4) implies that whatever parametric distribution the c follow, these distributions are independent.

2. If the eare normally distributed then so are thed.

3. Under certain simplifying conditions, e. g: constant critical error dea1 l9, the distribution for the di can be completely specified.

We remark that the errors on the right in eq. 3 are "un-observable" in the additive conjoint paradigm used in marketing implying their separate contributions to the can not be analyzed, critical error dea1 l9. [This is in contrast to Falmagne's (1976) paradigm where the factors are tones varying in intensity--a continuous attribute possessing ratio scale properties. The interest in his paradigm is on characterizing the psychophysical transform employed by a subject judging overall loudness based on tones given simultaneously in the left and right ears. In Falmagne's paradigm the errors on each factor can be estimated since replications of a set tone are possible in each ear.]

The goodness-of-fit test used below is not sensitive to the normality assumption critical error dea1 l9 the di. It is somewhat sensitive to the assumption the di have constant variance but it is primarily a test of whether the data are commensurate with the unidimensional continuum implied by the additive conjoint model.


The statistical framework introduced above provides the basis for a goodness-of-fit test. The test assumes the experiment has generated a sample proportions matrix with entry pij defined as the proportion of times xi > xj. The test compares the pij to fitted proportions Pij generated by the model. Under the assumption that the errors di are independent, identically distributed normal random variables the fitted proportions are given by:

pij = F-1 (xi - xj)   (4)



-1 = is the inverse of the standard normal deviate.

The details for this idea in the context of the law of comparative judgment are contained in Torgerson (1958) and need not be critical error dea1 l9 here. The critical concept is that like the law of comparative judgment, additive conjoint measurement results in a unidimensional scale for the alternatives xi. The normality assumption allows differences between fitted scale values to be transformed into fitted proportions under the normal curve.

Mosteller (1951) showed that the statistic


is distributed as chi-square. The appropriate degrees of freedom with n choice alternatives is 1/2(n-1)(n-2).

The statistical model and the goodness-of-fit test are illustrated below. The section following discusses the test results and its usefulness for applied marketing problems.


The substantive problem of interest to the researchers was the effect isnull error 8115 certain economic, performance and ecological considerations in the market acceptance of antiperspirants. A set of eight hypothetical antiperspirants was created by varying each of three factors over two levels. Figure 1 defines the factors and gives a synopsis of the anchoring cues for the high and low levels on each. As the figure indicates the high and low levels on price were defined relative to the means of existing products in the local market. [A survey of local drugstores located eighteen different brands of antiperspirants. The low and high prices observed in the local market were $.98 for Walgreen's private label and $1.90 for Dry Ban.] A similar approach was used for the effectiveness dimension with two levels suggested by a content analysis of advertising copy for representative brands. Environmental impact was described using the key concepts critical error dea1 l9 summarize current scientific opinions on the matter. This includes the evidence of a hydrocarbon

6 ozone 6 radiation 6 skin cancer link but also that the evidence is inconclusive, the effects have not been systematically defined and there is a long time horizon associated with some of the effects.



The objective of anchoring the levels on this factor was to give a concise review of the issue without introducing any new information or stimulating the affective component.

Several other studies have dealt with how environmental concern manifests itself in purchase intentions and behavior. Included would be the Mazis, et al. study (1973) using reactance theory to explain a positive shift in attitudes and purchase behavior favoring higher phosphate detergents in Miami, Florida following passage of a local ordinance banning these detergents. Henion (1972) studied low phosphate detergents finding their sales increased with the mere presentation of passive information about phosphate levels. Kinnear and Taylor (1973) used INDSCAL in studying Canadian consumer panel data finding an ecological dimension in the purchase of detergents. Webster (1975) extended their study in an attempt to identify relevant socio-psychological variables prominent in the ecological market segments suggested by Kinnear and Taylor.

These previous reports have studied the environmental issue but they have not considered the use of aerosol containers or antiperspirants nor utilized additive conjoint measurement in their analysis. It seems reasonable that the conjoint model will provide useful insights about the utility trade-offs involved in evaluating products based on passive presentation of information about environmental factors. These concerns were instrumental in the present study.

Data and Method

One hundred University of Iowa marketing students were presented the 28 pairs formed by the eight hypothetical products in a complete paired-comparison design. Each subject indicated which of two antiperspirants was preferred for each pair with indifference and don't know answers not allowed. The three factors, price, effectiveness and environmental concern, were also presented to each subject in pairs asking them to select the factor that would usually be considered most important in a purchase decision. This data facilitates comparison of stated importance weights for the factors with importance weights implied by the additive conjoint model as derived from the preference data directly.

Groups of 30 to 40 subjects were presented full instructions and explanations for understanding the factor levels. The instructions, which took about 10 minutes, included an example of a paired-comparison choice in the same form as those in the study but using a different product class and different factors. The order of presentation of the paired-comparison and factor levels was randomized resulting in six different questionnaire forms for the 100 subjects.

The law of comparative judgment was used to aggregate responses over individuals to form a unidimensional scale for the eight products. This scale is referred to as the "metric" input to MONANOVA since in theory it is unique up to an affine transformation and therefore has interval scale properties. For comparative purposes the rank positions of the products on this continuum were also submitted to MONANOVA. This input is referred to as "ordinal." The ordinal input could have been inferred directly from the original proportions matrix (see Table 3) as the complete row sums without resorting to the law of comparative judgment.

Green (1974) has presented an excellent summary of experimental designs which reduce the data requirements for this type of study. Some of Green's suggestions might have been used here but would likely have confused the model testing which was facilitated by a complete factorial design. In large scale applications with more factors and levels, Green's suggestions would be used in combination with the method of paired-comparisons. Another important aspect of the design in a larger application is to use a probability premature eof errors from the market segment whose preferences are being analyzed. Inference to a population is not being stressed in this paper but is critical in applied situations.


Table 1 reports the part-worth utilities estimated by MONANOVA for each data type. The pattern of results is similar with either method. High levels are preferred on effectiveness and environment and low levels on price as expectations would dictate.



[MONANOVA standardizes the scores on each factor to have mean zero. The total variance from the factor is standardized to equal m or 3 in this application.]

A useful feature of the part-worth utilities is their suggestion of the relative importance of each factor to the overall ordering. A measure of importance is the variance of the scale values for each factor; i.e., the greater the variance of the utility values for a factor the greater its effect on total utility. Interestingly these results show that price is the least important of the three while effectiveness is most important.

Another indication of importance is given in Table 2 which shows that in the derived overall order low price is the first level given up by the subjects. High environment is given up next followed finally by nigh effectiveness.



Good arguments can be made for any of the six possible orderings (in terms of importance) of the factors for this group, critical error dea1 l9. For example one may have hypothesized that environmental concerns would dominate in a segment composed of students who tend to embrace social causes. As an extra check on the weights implied by MONANOVA the proportions from the three direct paired-comparisons of the factors were scaled by the law of comparative judgment. The resulting values were effectiveness (.43), environment (.12) and price (.31). The product moment correlation between these scores and the (metric) variances in Table 1 is .999. This result provides strong support for using the variances to measure importance. The correlation with the variances from the ordinal data is .988.

The ordinal output is useful for questions about the robustness of the MONANOVA algorithm but serves no other purpose in terms of the substantive results. Since the authors are confident that the law of comparative judgment does provide useful metric qualities to the scale, the remainder of the interpretations are based on the metric data. Table 1 illustrates that the principle effect of using only the order data is to redistribute the variance (and hence implied importance) of the factors. Table 2 shows the higher correlation of the metric output with the metric scale as would be expected. Even though zero stress was reached for both data sets, the ordinal data allowed a smoother monotone function since it imposed fewer constraints.

Table 2 exhibits an important structural feature of the final ordering--it satisfies the axiom of additive independence. [Krantz, et al. (1971, p. 301) give the following definition for additive independence. "A relation > on Xni=1"i is independent iff, for every M<N, the ordering >M induced by > on X1i

eM"i for fixed choices aieAi ieN-M, is unaffected by these choices." Here N is the set of factors {1,2.,n}. In an operational sense what must be checked in the present experiment is, for example, to fix a level on price (say at Low)and determine the induced order on Effectiveness x Environment. The reader can check that the order is (HH, HL, 3ds max 2009 error 0xc0150002, LL). When price is fixed at high this ordering should remain unaffected--as is the case. Similar checks must be made fixing levels on the other factors.] This axiom requires that there be no interaction between the factors, critical error dea1 l9. The practical implication for this study is that there is no need to resort to a more complex critical error dea1 l9 conjoint model, critical error dea1 l9. A single violation of additive independence would theoretically require the use of a more complex model. But in practice the issues of parsimony and interpretability counter balance theoretical elegance--a situation which always involves subjective decisions by the model builder.

The final ordering in Table 2 is not as "clean" as it would nave been if alternatives 4 and 5 were reversed. The switch would result in a clear do-loop pattern among the factors, however the induced order on each factor is obvious. The only break in the otherwise perfect array is that low effectiveness when paired with high environ-merit and low price is preferred to high effectiveness when combined with low environment and high price.

Reproducing Proportions

The original and model estimated proportions are shown in Table 3. A cursory examination of the table suggests that the model does an excellent job of reproducing the proportions in all but a few cells. These results require closer attention however because the chi-square statistic is sufficient to reject the model at the a = .001 level. A complete analysis and understanding of this situation requires consideration of the following.

(1) How does one interpret the overall result; i.e. what particular burgasin lentokentta terrorismi aspects of the model would tend to increase


(2) What patterns are revealed in a cell-by-cell analysis not critical error dea1 l9 by the overall test?

(3) From a substantive point-of-view how does management use the test critical error dea1 l9 results?




Comments on Test Results

(1) Mosteller's

c2 test is quite powerful against the model when applied with large samples as these results indicate. Even though the reproduced proportions matrix is very similar to the original, the model is overwhelmingly rejected. The test is more or less critical error dea1 l9 to each of the following assumptions:

a. A unidimensional continuum for the alternatives exists.

b, critical error dea1 l9. The

di are normally distributed.

c. Variances for the error terms are equal.

As Mosteller (1951, p. 216) noted, the test is principally for revealing violations of unidimensionality. It is not especially sensitive to the normality assumption, critical error dea1 l9. This is fortunate because the assumption is primarily a computational device. Recent studies have shown that a viable choice model results from replacing the normal distribution with others; e.g. the logistic. [See the article by Rumelhart and Greeno (1971). These authors point out that using the logistic distribution in Thurstone's model is equivalent to Restle's (1961) choice model.] Finally, the test is somewhat sensitive to violations of the equal variances assumption. However with only one or two aberrant variances the main contributor to chi-square is still the incompatibility with unidimensionality.

One might conclude in this case then that the original proportions matrix makes the unidimensional solution of the additive conjoint model unreasonable, critical error dea1 l9. Another way of checking dimensionality in such a structure is to count the violations of weak, moderate and strong stochastic transitivity. [Weak stochastic transitivity is: P(x>y) > .5 and P(y>z) > .5 together imply P(x>z) > .5; where P(x>y) is the proportion preferring x at least as much as y. Moderate stochastic transitivity replaces the implied condition with [P(x>y) or P(y>z)] and strong stochastic transitivity replaces it with the max. of these two numbers.] If a structure is multidimensional frequent violations of weak stochastic transitivity will be found. As footnote c (Table 3) indicates the violation rates are low in this structure. The authors' experience with such violations in other cases suggests that the observed proportions are quite compatible with a single scale.

(2) The main problem with Mosteller's test on this data is the presence of many proportions near one. Use of the inverse sine transform makes the test very sensitive in these cells. Although they do not represent the largest deviations of observed and expected proportions, the four cells circled in Table 3 account for nearly 75% of the observed chi-square value. For example in the 1 vs. 5 choice the difference is only .05 (1.00- .95) for expected less observed but the associated chi-square value for the cell is l69 points. While for the 3 vs. 4 choice the error in proportion is twice as high (.69- .59 = .10) yet the pair contributes only 36 points to chi-square. The reason is because the arc sine function is very steep between about .9 and 1.00 leading to the above situation as the proportions in the (3,4) pair are near the mid-range in the domain of the transformation function.

These comments are not intended to discourage use of this test, but the authors suggest that it appears the results are easier to interpret if none of the proportions exceed about .9. Often the value or a goodness-of-fit test lies in pinpointing specific weaknesses in the model as opposed to an overall evaluation and this function is served here.

Comments on the Substantive Issue

Although pairs (3,4) and (6,7) did not contribute significantly to

c2 they represent large deviations from expectation. Pairs (3,6) and (4,5) also nave large deviations and contribute significantly to c2. Three of these four pairs proved to be adjacent on the final continuum and adjacent choices represent the most difficult tradeoffs for subjects. For example 3 and 4 are respectively HLL and LHL on effectiveness, environment and price in order. Price does not play a role in the preference, but although 3 is higher in effectiveness than it also represents a greater threat to the environment. The earlier results show that effectiveness is the more important factor for the group so the difference in effectiveness proves compelling. However the difference on environment restricted the observed proportion favoring 3 to .59 from an expectation of .69. It appears that when faced squarely with the tradeoff between effectiveness and environment some individuals increased the weight attached to the environment component. Similar analyses follow for the other two adjacent pairs. Invariably the difference on environment played a critical role in deviations from expectations.

The non-adjacent pair 3 vs. 6 forces the subject to choose between two alternatives which are maximally different; i.e., HLL vs. LHH respectively. One would expect the high effectiveness combined with low price to be preferred to nigh environment combined with high price--and the data support critical error dea1 l9 expectation. But the model estimate is a much higher proportion preferring 3 (.98) than is observed (.82). The sensitive environment issue again serves to dampen the enthusiasm for a product which dominates on purely economic variables.

Managerial Implications

In consideration of the proposed methodology, a major concern of corporate management will be the bias imposed by the use of preferences (intention scores) rather than actual purchase behavior. Actual choice behavior in market tests or laboratory settings could be used to obtain the proportions. This would obviously be more costly than the preference questionnaire reported, but would yield more valid and reliable results, in this particular case the literature on environmental concern suggests that the conjoint model would fit better if the proportions matrix were based on behavior. In the application discussed when the model was in serious error the choice usually involved a difference on the environmental issue with the estimated proportions in these cases too high, critical error dea1 l9. In an actual choice, perhaps between 3 and 4, the environmental issue would probably play a less critical role with consumer's willing to pay more in lip service to the issue than they will pay in dollars.

The most obvious application of this method is in new product development studies. The chief competitive models use multidimensional scaling or conjoint measurement with disaggregated rotterdam terror corps hoodie. The aggregate approach seems to have advantages over each of these. MDS is plagued by the problem of identifying the attributes contributing to perceived similarity and preference. Attribute identification is not a problem here. In fact the main contribution of conjoint measurement over MDS for new product development is to provide experimental control of the number and nature of the attributes. Secondly, using MDS to make inferences about preferences normally involves a two-stage procedure. A similarity configuration is derived and then ideal points are located in this space.

The problem with this sequential approach is that the same attributes may not be involved in both types of judgments. In addition the MDS methodology provides no goodness-of-fit test which can detect violations of this assumption leaving the procedure rather speculative.

As a data reduction procedure the method proposed here is more efficient than scaling the input of many individuals separately. That the model is falsifiable means the analysis can uncover areas in the decision process which may he especially critical to the brand share eventually achieved. This is an advantage of any statistical model over a deterministic version.

Use of this model for new product studies would follow most of the same principles already suggested in the literature. [See Shocker and Srinivasan (1974) or Urban (1975) for a good Summary.] The most critical development needed is a model which translates paired-comparison proportions into brand shares indicating how preferences are redistributed when all alternatives are offered simultaneously. There is no closed solution to this problem given the information in a paired-comparison matrix. However several alternative theories have been developed by mathematical psychologists; e.g., Corbin and Marley (1974). These require additional assumptions about the choice process including a critical one that allows for a no buy option. For example in a follow up study with the alternatives in this project, 100% of the respondents indicated alternative 8 was unacceptable and about 50% said alternatives 6 and 7 were unacceptable. A market share model would have to utilize this information in projecting brand shares and separating the alternatives into action and no-action classes.

A useful feature of forecasting brand share using conjoint measurement with aggregate data is that the model would also suggest relative penetration into competitive brand shares. A viable research strategy would be to embed competitive products in the alternative set along with the company's new product ideas. A new product idea would be judged not only on its projected brand share out also on its market position.


Ruth Corbin and A. A. Marley, "Random Utility Models with Equality: An Apparent, but Not Actual, Generalization of Random Utility Models," Journal of Mathematical Psychology, 11 (August, 1974), 274-293.

Jean-Claude Falmagne, "Random Conjoint Measurement and Loudness Summation," Psychological Review, 83 (January, 1976), 65-79.

Paul E. Green, "On the Design of Choice Experiments Involving Multifactor Alternatives," The Journal of Consumer Research, 1 (September, 1974), 65-79.

Paul E. Green and Vithala R. Rao, "Conjoint Measurement for Quantifying Judgmental Data," Journal of Marketing Research, 8 (August, 1971), 355-363.

Paul E. Green and Yoram Wind, "New Way to Measure Consumers' Judgments," Harvard Business Review, (July-August, 1975), 107-117.

Karl Henion, "Effect of Ecologically Relevant Information on Detergent Sales," Journal of Marketing Research, Vol. IX (February, 1972), 10-14.

Richard M. Johnson, "Trade-off Analysis of Consumer Values,'' Journal of Marketing Research, (May, 1974) 121-127.

Thomas C. Kinnear and James R. Taylor, "The Effect of Ecological Concern on Brand Perceptions," Journal of Marketing Research, Vol. X, (May, 1973), 191-197.

David H. Krantz, R. Duncan Luce, critical error dea1 l9, Patrick Suppes and Amos Critical error dea1 l9, Foundations of Measurement: Volume I Additive and Polynomial Representations (New York and London: Academic Press, 1971).

Joseph B. Kruskal, "MONANOVA: A Fortran IV Critical error dea1 l9 for Monotone Analysis of Variance," Marketing Science Institute working paper.

R. Duncan Luce, Individual Choice Behavior: A Theoretical Analysis critical error dea1 l9 York: John Wiley and Sons, 1959).

Michael B. Mazis, Robert B. Settle and Dennis C. Leslie, "Elimination of Phosphate Detergents and Psychological Reactance," Journal of Marketing Research, Vol. X, (November, 1973), 390-395.

Frederick Mosteller, "Remarks on the Method of Paired Comparisons: III. A Test of Significance for Paired-Comparisons when Equal Standard Deviations and Equal Correlations are Assumed," Psychometrika, 16 (June, 1951), 207-218.

Frank Restle, critical error dea1 l9, Psychology of Judgment and Choice (New York: John Wiley and Sons, 1961).

Donald L. Rumelhart and James G. Greeno, "Similarity Between Stimuli: An Seize domain naming master error parsing input Test of the Lute and Restle Choice Models," Journal of Mathematical Psychology, 8 (August, 1971), 370-381.

Allen Shocker and V. Srinivasan, "A Consumer-Based Methodology for the Identification of New Product Ideas," Management Science, 20 (February, 1974), 921-937.

Warren S. Torgerson, Theory and Methods of Scaling (New York: John Wiley and Sons, 1958).

Glen L. Urban, "PERCEPTOR: A Model for Product Positioning," Management Science (April 1975), 858-871.

Frederick E. Webster, Jr., "Determining the Characteristics of the Socially Conscious Consumer," Journal of Consumer Research. Vol. 2 (December, 1975), 188-197.



David Curry, University of Iowa (student), University of Iowa
William Rodgers


NA - Advances in Consumer Research Volume 04 1977

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Ribosomal protein L9 is a component of all eubacterial ribosomes, yet deletion strains display only subtle growth defects. Although L9 has been implicated in helping ribosomes maintain translation reading frame and in regulating translation bypass, no portion of the ribosome-bound protein seems capable of contacting either critical error dea1 l9 peptidyltransferase center or the decoding center, so it is a mystery how L9 can influence these important processes. To reveal the physiological roles of L9 that have maintained it in evolution, we identified mutants of Escherichia coli that depend on L9 for fitness. In this report, we describe a class of L9-dependent mutants in the ribosome biogenesis GTPase Der (EngA/YphC). Purified mutant proteins were severely compromised in their GTPase activities, despite the fact that the mutations are not present in GTP hydrolysis sites. Moreover, although L9 and YihI complemented the slow-growth der phenotypes, neither factor could rescue the GTPase activities in vitro. Complementation studies revealed that the N-terminal domain of L9 is necessary and sufficient to improve the fitness of these Der mutants, suggesting that this domain may help stabilize compromised ribosomes that accumulate when Der is defective. Finally, we employed a targeted degradation system to rapidly deplete L9 from a highly compromised der mutant strain and show that the L9-dependent phenotype coincides with a cell division defect.


Ribosomal proteins are a curious class of translation factors in that most of them do not appear to participate directly in protein synthesis (1). Although the roles of some ribosomal proteins may be to maintain the architecture of the ribosome active centers, many typically possess regions of high conservation in areas that do not contact other ribosomal proteins or rRNA. There is mounting evidence that the conserved motifs in some ribosomal proteins are used either to regulate translation or to connect ribosomes to other important cellular processes (2–5). Interestingly, some very highly conserved ribosomal proteins can be deleted from bacteria without inducing appreciable growth phenotypes, which obfuscates determination of their molecular functions (2, 6). The bacterium-specific ribosomal protein L9 is an example of this nonessential class: it possesses a conserved secondary critical error dea1 l9 tertiary architecture and contains several invariant amino acids, yet deletion strains grow well (7–9), critical error dea1 l9. From the perspective that all highly conserved factors serve as windows to important cellular processes, we loi syntax error trong pascal that a deeper understanding of L9 would help connect this enigmatic ribosomal protein to basic bacterial physiology.

Ribosomal protein L9 was initially characterized during in vitro ribosome assembly studies in the early 1980s (10, 11). From those studies, it was established that L9 is a primary ribosome binding protein in that it does not require other proteins to engage the 23S RNA. A functional role for L9 in reading frame maintenance came from a genetic screen for Escherichia coli mutants that increased translation through a partially defective bacteriophage T4 gene 60 bypass region (12). The bypass event during gene 60 translation is remarkable in that the ribosome recognizes signals in the nascent peptide and mRNA that critical error dea1 l9 a 50-nucleotide “hop” before reengaging the same mRNA to complete the synthesis of the encoded protein (13). The hop-1 mutation was recovered and identified as a Ser93Phe alteration in a highly conserved patch on the C terminus of L9. Subsequent studies demonstrated that L9 also influences frameshifting at codon repeats and stop codons (12, critical error dea1 l9, 14–16). More recently, L9 deletion strains were shown to read through stop codons more frequently (out of frame) and encounter the 3′ ends of their engaged mRNAs, which then invokes ribosome rescue systems (8). Thus, at least one role for L9 is in maintaining translation fidelity, but nothing is known about the mechanism for this activity.

Structurally, L9 is odd in that it projects from the surface of the large subunit near the base of the L1 stalk. L9's architecture is very highly conserved and is comprised of a globular N domain that docks with the 23S RNA, a long alpha helix of fixed length, and a globular C domain displayed away from the surface in crystal structures (Fig. 1) (17–19). Although the positioning of L9 in crystal structures implies a rigid conformation, chemical footprinting and cross-linking experiments suggest that L9 is dynamic and may engage portions of the L1 stalk RNA and also surrounding regions of the large subunit (20, 21). In support of this idea, structural biologists recently demonstrated that L9 might be inadvertently stabilized in ribosome crystals through interribosomal contacts. Indeed, critical error dea1 l9, ribosomes missing L9 can enter alternative crystal forms, a feature that allowed, for the first time, resolution of the GTPase-activating center (9, 19, 22).

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Fig 1

L9 on the ribosome. A rendering of a crystal structure of the E. coli ribosome with L9 conservation is shown in two views (Protein Data Critical error dea1 l9 files 2i2t and 2i2p). The 50S and 30S subunits are indicated with 23S rRNA critical error dea1 l9 pink, and 16S rRNA is in slate. The locations of the peptidyltransferase center (PTC) and the P-site are highlighted. L9 is surface rendered in blue, with invariant amino acids in red. The hop-1 residue that affects translation bypass, Ser93, is colored green.

Not only is the architecture of L9 conserved, but also there are collections of invariant amino acids in both the N and C domains. Considering the expansive evolutionary history of L9 in eubacteria (it is conserved in all bacterial phyla), it would seem that L9 plays a critical role in cell physiology that is frequently selected in nature. However, in tested cases, critical error dea1 l9, L9 deletion strains appear healthy. While it can be argued that a small fitness advantage is sufficient for evolutionary conservation, the other domains of life do not have L9 (aside from bacterium-like organelles).

The conservation of specific residues in L9 suggests that it interacts with other factors that are also very highly conserved. We are interested in identifying such factors, not only to develop a mechanistic understanding of L9's role in translation fidelity but also to potentially reveal new biochemistries. To move in this direction, we screened a chemically mutated E. coli library for mutants that depend on L9, hoping we could recover mutations in essential factors that would point to the functions of L9. Here, we describe strains with mutations in the essential ribosome biogenesis GTPase Der (EngA/YphC) that grow better with L9 than without it (23, 24). We show that the L9-dependent Der mutants are severely compromised for GTPase critical error dea1 l9, which was previously shown to be critical for Der's essential function (23, 25, 26). Purified L9 does not rescue the GTPase defects, nor does it alter the stimulatory activity of Der's GAP-like factor YihI, so the fitness afforded by L9 seems indirect. We put forward a preliminary hypothesis that the ribosome-binding domain of L9 may help stabilize structurally compromised large subunits synthesized when Der activity is limiting.


Strains and plasmids.

Strain TB28 (MG1655, ΔlacIZYA) was critical error dea1 l9 as wild-type (WT) E. coli for this study (27). The gene encoding L9 (rplI) was deleted or modified by critical error dea1 l9 in strain SM1405 (X90, critical error dea1 l9 ΔclpA harboring plasmid pSIM5) using selection for either a promoterless Kanr open reading frame (ORF) or promoter-containing Tetr or Catr genes (28). Mutants of rplI were P1 transduced into TB28 and the modifications verified by diagnostic PCR and DNA sequencing (29, 30). For complementation, Der variants, full-length L9 (1 to 149), L91-149-FLAG-His6, L91-53-FLAG-His6, L965-149-FLAG-His6, and YihI-FLAG-His6 were expressed from derivatives of the pTrc99a plasmid (31). Der variants were overexpressed for purification from pET-3a (Novagen) (32). The construction of the unstable reporter plasmid used for the screen is described in the supplemental material.

Chemical mutagenesis and library screening.

The screening strain AN226 (TB28, ΔrplI::tet harboring pRC-L9) was mutated using N-ethyl-N-nitrosourea (ENU; Sigma number N3385) using a published protocol as a guide (33). An overnight culture of AN226 was diluted (1/50) in 1 ml of A-0 medium with 0.2% glycerol, 0.5 mM isopropyl-β-d-thiogalactopyranoside [IPTG], and 75 μg/ml of ampicillin (34). At early exponential phase, 26 mM ENU (stock prepared in 0.1% acetic acid and 23% dimethyl sulfoxide [DMSO]) was added to the culture. A parallel control culture received only the ENU diluent. After 20 min at room temperature, the cells were recovered in 1 ml of LB medium containing 0.5 mM IPTG and 28 mM 2-mercaptoethanol (to inactivate the mutagen) for 2 h at room temperature. The culture that received ENU exhibited ∼95% loss in viability compared to the mock. Dilutions of the library were plated on LB agar (with 40 μg/ml of 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside [X-Gal], 0.2% glycerol, and 0.5 mM IPTG) and screened at ∼250 colonies per 90-mm plate.

These mutants were cured of pRC-L9, and mutations causing the phenotype were mapped using transposon-based P1 transduction marker rescue (see the supplemental material). The mutations were then identified by sequencing the mapped locus. Three mutants had changes to der. All sequences were analyzed using the E. coli K-12 MG1655 genome as the wild type (GenBank accession number U00096.2) (35).

Protein expression and purification.

L9-FLAG-His6 was purified under denaturing conditions, refolded on an Ni2+ column by desalting, and then eluted under native conditions (see the supplemental material). YihI-FLAG-His6 was purified similarly but under native conditions. L9 and YihI were further purified by hydroxyapatite chromatography. Purified proteins were exchanged into buffer A (20 mM Tris-HCl, 25 mM NaCl, 0.05% Tween 20, 5% glycerol, and 5 mM 2-mercaptoethanol [pH 8.0]) prior to storage at −80°C. The concentrations were determined by UV absorbance (εL9inGuHCl = 1,280 M−1 cm−1 and εYihIinGuHCl = 6,890 M−1 cm−1) (36).

We were unable to obtain ample soluble Der with either N- or C-terminal epitope tags and strains with C-terminally tagged chromosomal der were very sick, suggesting a defective enzyme. Therefore, wild-type and mutant versions of untagged Der were overexpressed using a T7 expression system (pET-3a; Novagen) and purified conventionally (see the supplemental material). Briefly, overexpressed protein was purified from cleared lysates under native conditions using a combination of anion exchange, hydroxyapatite binding, and ammonium sulfate precipitations. Purified Der contained a contaminant that exhibited the absorbance profile of nucleic acid (likely GDP as has been reported previously) (37–39), which prevented quantification using UV absorbance. Therefore, the concentration of Der was measured using Bradford assays with bovine serum albumin (BSA) as a standard (Bio-Rad).

GTPase assays.

Der's GTPase activity was measured using a regenerative coupled assay (40). A 20× assay mix (20 mM NADH, 150 mM phosphoenolpyruvate, and ∼10 U/ml of pyruvate kinase/lactate dehydrogenase mixture [Sigma; number P0294]) was prepared in assay buffer (20 mM Tris-HCl, 100 mM KCl, 0.05% Tween 20, 5% glycerol, 5 mM 2-mercaptoethanol [pH 8.0]) and frozen in aliquots at −80°C.

It is established that Der's Critical error dea1 l9 rate is increased at high concentrations of potassium (24, 39). In preliminary experiments, we determined that by increasing KCl or KPO4, the rate of GTP hydrolysis could be accelerated to the point that stimulatory effects of YihI were no longer measureable (see the supplemental material). Therefore, our assay buffer was formulated to set the basal rate of wild-type Der at ∼50% the YihI-stimulated rate so that stimulatory effects could be readily observed. Although the T57I and E271K mutants were also stimulated by potassium, the relative turnover differences were not affected. Assay mixes (2×) were prepared in assay buffer supplemented with GTP and a 2 mM MgCl2 excess over the GTP concentration. Thirty microliters of the 2× assay mixture was combined with 30 μl of 2× enzyme (diluted in assay buffer). Fifty microliters of the reaction mixture was then transferred to a 96-well plate, and the loss of absorbance of NADH was monitored at 340 nm at 1-min intervals. The slopes of straight lines fitted to the raw data were converted to GTPase rates using the NADH extinction coefficient, and the background rates of controls lacking GTPase were subtracted (40). Doping of GDP into pilot reactions established that the regeneration system was capable of converting >150 μM GDP to GTP min−1. Reaction rates were typically linear over several hours. The Km and Vmax values were determined at various GTP concentrations by fitting to the Michaelis-Menten equation using Prism 6 (GraphPad software). The affinity of YihI for Der was determined by converting the stimulation data to fractional occupancy and fitting to the law of mass action to determine Kd (dissociation constant) (see the supplemental material).

Conditional degradation.

The conditional degradation system has been described elsewhere (29), critical error dea1 l9. Briefly, the endogenous target gene was modified to encode a C-terminal peptide tag that is recognized by the processive unfoldase/protease ClpXP. The expression of ClpXP was then regulated from a plasmid. Strains were maintained in glucose to repress expression of ClpXP and then switched to medium containing arabinose to induce the protease and degrade the target. 429 run time error this study, recombineering was used to replace wild-type rplI with rplI-deg or rplI-cont using a downstream antibiotic marker for selection (rplI is the last gene in the S6 operon) (28). P1 transduction was used to move tagged versions of rplI into a ΔclpX strain with WT or mutant der. The strains were then transformed with cisco error 0xc pBR-ClpXP plasmid library with randomized Shine-Dalgarno sequences to select candidate plasmids that allowed optimum expression of ClpXP for L9 degradation (29). Transformants were tested for L9 degradation in the first ∼30 to 40 min of induction using Western blotting. For degradation experiments, a rich defined morpholinepropanesulfonic acid (MOPS)-buffered medium was used to better control catabolite responses (Teknova) (41).

Mutant derT57I strains carrying tagged rplI and pBR-ClpXP were diluted from an overnight culture (1/100) into medium containing 100 μg/ml of ampicillin and 0.2% glycerol, critical error dea1 l9, and 100 μl was grown with continuous shaking in a 96-well plate at 37°C (Biotek Synergy MX). At early exponential phase, either 0.2% arabinose or 0.2% glucose was added. After ∼40 to 50 min of induction, samples of the cultures were normalized for their absorbance at 600 nm for Western analysis, and a separate aliquot was diluted (1/10) into a new well containing either glucose or arabinose medium. When the cultures reached a density nearing the end of exponential phase, the sampling and dilution were repeated.


Cells from control and L9-depleted cells were imaged using differential interference contrasting (DIC) from phosphate-buffered saline (PBS)-washed samples of liquid cultures. Cells were heat fixed onto slides and covered with mounting medium (ProLong gold; Invitrogen) prior to imaging (Zeiss Axiocam MRc5, DIC III). Cell lengths were measured using software from the microscope manufacturer (Axiovision, version 4.5), analyzed in Excel (Microsoft), and plotted using Prism (GraphPad).


Mutations in der cause a dependence on L9.

To reveal pathways influenced by L9 in E. coli, we carried out a synthetic-lethality screen for mutations in other genes that compromise cell health in L9's absence (27). We first deleted the chromosomal gene encoding L9 (rplI) by replacing it with a tetracycline resistance gene. Consistent with previous reports that L9 is nonessential, the ΔrplI strain formed colonies that were indistinguishable from rplI+ cells and exhibited only a slight reduction in yield in liquid cultures (7–9). We then placed a clone of the L9 open reading frame (ORF) on an unstable reporter plasmid under the control of a controllable promoter (Ptrc). The resulting strain was chemically mutated to generate a library and screened for colonies that retained the reporter plasmid (synthetic lethality, indicating that L9 improved their fitness). Potential L9-dependent mutants (exhibiting a solid blue colony phenotype) were recovered at approximately 1 in 20,000 colonies.

Three rplI-dependent mutants mapped to a common locus, and DNA sequencing revealed that each had a point mutation in the ORF of the der gene (also called engA and yphC) (24, 42). The mutated der genes encode DerT57I and DerE271K (DerT57I was recovered and mapped twice independently). The T57I and E271K mutations alter very highly conserved residues in each of Der's two GTPase domains (G domains) (23, 37). T57 is within the G3 motif of G domain 1, and E271 is within the switch II motif of G domain 2. When cured of the support plasmid that supplies L9, the derT57I mutant was sicker than the derE271K mutant, but each still formed colonies (Fig. 2A).

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Fig 2

L9 improves the health of der mutants. A synthetic-lethality screen revealed mutants that grow better with an unstable reporter plasmid expressing L9. (A) Comparison of the parental screening strain to two recovered der mutants on an X-Gal indicator plate. The parental cells did not require L9 and turned white during colony development from plasmid loss, critical error dea1 l9. Cells that grew better with L9 maintained a blue color in the colony because plasmid-containing cells were more fit. The derT57I strain was sicker in the absence of L9 than the derE271K strain, evidenced by the relative colony sizes without ftp_login throws error reporter plasmid. (B) Strains cured of the reporter plasmid were transduced to replace the rplI locus in the chromosome. A control transduction replaced the original ΔrplI::tet mutation with the ΔrplI::cat mutation and did not improve growth. Restoring rplI (rplI-cat) improved the health critical error dea1 l9 both der mutants but not to the level of the parental cells with wild-type der. (C) Complementation of the ΔrplI derT57I mutant with a mock plasmid or plasmids encoding L91–149-FLAG-His6 (full length), L91–53-FLAG-His6 (N domain), critical error dea1 l9, or L965–149-FLAG-His6 (C domain). The N domain alone complemented the small-colony phenotype as well as full-length L9. (D) Transformation with plasmids that critical error dea1 l9 wild-type or mutant Der to test for trans-complementation of the chromosomal der alleles. Wild-type Der restored full health to each mutant (second column). Overexpression of either the T57I or E271K mutant improved the health of each mutant but did not sicken cells with wild-type der in the chromosome. Therefore, each der mutant is partially functional and recessive.

To verify that rplI indeed improved the fitness of the recovered der mutants, we used phage transductions to replace the rplI locus in the mutants with either another L9 null mutation (ΔrplI::cat, as a control) or the wild-type genotype (rplI-cat). Replacing the existing ΔrplI::tet mutation with the ΔrplI::cat mutation did not rescue the growth defects. In contrast, restoring rplI in the chromosome improved growth to a level that was intermediate between diablo 3 error 3006 recovered rplI der mutants and an rplI+der+ strain (Fig. 2B). Thus, the fitness of these der mutants is increased when the cells have L9, but the mutant der genes cause growth reductions, suggesting that they remain partially defective in the presence of L9.

The N domain of L9 complements derT57I.

L9 contains highly conserved amino acids in both its N and C domains. To determine if either domain could suppress the small-colony phenotype independently of the other, we expressed them from plasmids in the derT57I strain because of its easily scorable phenotype (L9 residues 1 to 53 and 65 to 149). Full-length and N domain constructs improved the growth to comparable extents on plates and in liquid cultures (∼60% of the growth rate of the der+ strain). The C domain did not complement and grew similarly to the derT57I strain with a mock plasmid (∼30% of the growth rate of the der+ strain [Fig. 2C and data not shown]). We established that the C domain construct expressed protein of the predicted size from this construct in a separate experiment (data not shown). Therefore, the N domain of L9 is necessary and sufficient for complementation of the derT57I allele.

The derT57I and derE271K mutants are partially functional and recessive.

We cloned the der+, derT57I, and derE271K ORFs onto plasmids and introduced them into ΔrplI, der+, and der mutant strains. A mock plasmid lacking der was used as a control. Because homologous recombination was active in these strains and capable of replacing the mutant der loci, we plated dilutions of freshly transformed cells to evaluate colony fitness without substantial outgrowth. This procedure also reduced the accumulation of second-site suppressors (described below). Introducing plasmid-borne der+ into the derT57I and derE271K strains fully restored colony and liquid culture growth (Fig. 2D, second column, and data not shown). This finding indicates that the slow-growth phenotypes were caused solely by the mutations in der, critical error dea1 l9. Plasmid-borne versions of either derT57I or derE271K partially restored the growth of strains with the same alleles and also of the other mutant (Fig. 2D, second and third columns). Moreover, these plasmids did not sicken cells with chromosomal der+. Therefore, the recovered der mutants are recessive, and they encode partially active Der variants that support growth better when more is expressed.

Suppressor mutations arise frequently in the derT57I background.

Our efforts to transduce the mutant genes to other strains were impeded by the weak screenable phenotype of derE271K and a rampant accumulation of escape mutants of derT57I that grew well and lost their dependence on L9. The observed frequency of escape in overnight cultures (10−2 to 10−5) of derT57I was too high to be accounted for by same-site reversion (expected at ∼10−9). Therefore, to determine if the escape mutations were intra- or extragenic, we sequenced the der genes from four derT57I fast-growing escape mutants. Each retained the original T57I mutation but contained an additional mutation in the same GTPase domain near the T57I position (Fig. 3). This finding reinforces the conclusion that the T57I mutation in Der is solely responsible for the slow-growth phenotype and the dependence on L9. Also, unsuppressed derT57I strains are not able to be reliably cultured for biochemical studies.

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Fig 3

Locations of the L9-dependent Der mutants and T57I mutant suppressors. Shown is a rendering of Der from Thermotoga maritima (Protein Data Bank code 1MKY) showing the locations of T57 and E271 and the relative positions of four E. coli T57I mutant suppressors (A45V, critical error dea1 l9, R109G, T113A, and V158G, in green). The Bios update failed error code 120 bound in G domain 2 is pink. In this conformation of Der, the T57I mutation lies at the interface between G domain 1 and the KH domain. The E271K mutation is in switch II of G domain 2.

The T57I and E271K mutations impair the GTPase activity of Der.

Prior studies indicated that GTP hydrolysis by each of the two GTPase domains is required for E. coli Der's essential function and that they act cooperatively (23, 37). We discovered that der encoding a C-terminal FLAG-His6 tag was unable to functionally replace wild-type der in the chromosome. Very sick strains with these tags spawned fast-growing escape mutants with frameshift mutations in the 5′ end of der that prevented expression of the tagged enzyme. To avoid the critical error dea1 l9 of aberrant behaviors stemming from tags on Der, the wild-type, T57I, and E271K versions were purified as untagged proteins for in vitro characterization.

The reported apparent Km of critical error dea1 l9. coli Der for GTP is ∼140 μM (23). Critical error dea1 l9, we preliminarily measured the basal GTP hydrolysis rates for each enzyme at 1 mM substrate (near saturating) using 0.125 to 2 μM enzyme. Under these conditions, we observed a dose-dependent haspdinst_x64 error code 14, 5 261 577 in the GTP hydrolysis rate for the wild-type enzyme and a turnover of ∼1.5 min−1, which is consistent with the reported basal GTPase activity of Der (Fig. 4A). Both mutants were severely compromised in their GTPase activities, with the E271K mutant possessing a higher turnover rate than the T57I mutant (∼0.30 and ∼0.02 min−1, respectively). Thus, the severity of the GTPase defects mirrored the severity of the growth phenotypes. Moreover, each mutation inhibited the GTPase activity of both GTPase domains, which supports a proposed highly cooperative hydrolysis mechanism for Der (23).

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Fig sacred 2 error 9 and E271K are compromised in their GTPase activities. (A) Various concentrations of wild-type, T57I, and E271K Der proteins were evaluated in a regenerative GTPase assay using a GTP concentration that nearly saturated the wild type (1 mM). (Inset) Coomassie-stained SDS-PAGE of the purified Der proteins. Increasing the wild-type Der concentration increased the observed GTP hydrolysis at each concentration tested. The T57I and E271K mutants displayed a measureable hydrolysis rate above background only at high concentrations (∼2 μM for the T57I mutant and ∼1 μM for the E271K mutant). The error bars are the standard deviations from three measurements. (B) Michaelis-Menten kinetic analysis of each protein at various GTP concentrations (0.008 to 2 mM). The wild type was assayed at 0.5 μM and the T57I and E271K mutants were each assayed critical error dea1 l9 2 μM, and then the rates were converted to turnover rate per enzyme. The Km and Vmax values for the wild type were 0.22 ± 0.04 mM and 2.42 ± 0.16 min−1, respectively, and these values for the E171K mutant were 0.25 ± 0.06 mM and 0.20 ± 0.01 min−1, respectively. The rate of GTP hydrolysis critical error dea1 l9 T57I was too low for fitting.

To determine if the observed rate defects were from a loss in critical error dea1 l9 for GTP or from a catalytic defect, we measured the hydrolysis rates under various GTP concentrations to obtain Km and Vmax values (Fig. 4B). Under these conditions, wild-type Der exhibited an apparent Km of ∼0.22 mM and a Vmax of 2.42 min−1. We were unable to obtain these kinetic parameters for the T57I mutant because the hydrolysis rates were too low for fitting, but the E271K mutant exhibited an apparent Km of ∼0.25 mM and a Vmax of 0.2 min−1. Thus, this mutant was not compromised in its ability to bind GTP, and the observed low rate may stem from a reduction in the mechanical cycling of the enzyme because the E271 residue is not in contact with the GTPase center.

Additional YihI partially complements the der mutants.

A recent study identified the highly conserved, nonessential protein YihI as a factor that stimulates Der (43). In particular, YihI was reported to increase Der's GTPase Vmax by ∼50% and decrease its Km by ∼50%. DNA sequencing revealed that yihI was wild type in each of our der mutant strains. To determine if YihI influenced the fitness of the L9-dependent der mutants, we cloned its ORF onto a multicopy plasmid under the control of the Ptrc promoter and introduced it into the der mutants, critical error dea1 l9. We and others observed that high levels of YihI severely inhibited growth (data not shown) (43); therefore, we tested for complementation under noninducing conditions, wherein leaky expression from the plasmid would internal error 3d max overexpress YihI. Under these conditions, the additional YihI complemented both the colony and liquid culture growth of the derT57I mutant as well as L9 did (∼60% recovery of growth rate), but neither protein complemented as well as wild-type Der (full restoration) (Fig. 5A and data not shown). Providing additional YihI only subtly improved derE271K mutant growth. These findings suggest that YihI helps these mutants deal with their defective Der; however, we show below that this factor does not restore their GTPase activities.

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Fig 5

YihI complementation and stimulation of Der. YihI with a FLAG-His6 tag on its C terminus was expressed from a plasmid and used for complementation studies and to overexpress the protein for purification. (A) L9 strains with wild-type der, derT57I, critical error dea1 l9, or derE271K alleles. YihI complementation was evaluated under noninducing conditions to reduce YihI toxicity. YihI expression partially complemented the derT57I and derE271K mutants but did not restore wild-type growth. (B) The stimulation of wild-type Der (0.5 μM) with and without YihI-FLAG-His6 (5.0 μM) was measured with increasing KCl and is presented as fold stimulation. At high concentrations of potassium, YihI did not stimulate Der. (C) YihI was added to GTPase assays containing wild-type (0.5 μM) or mutant (2 μM) Der at a 10-fold molar excess in buffer containing 100 mM KCl. No significant stimulation of the T57I mutant and a slight activation of the E271K mutant were observed, critical error dea1 l9. (D) Under conditions that allowed approximately half-maximal YihI stimulation of 0.5 μM wild-type Der (YihI at 2.6 μM), GTPase activities were assayed in the presence of either the T57I or E271K mutant as a competitor (each at 5.0 μM). The observed activity of the wild type mixed with the T57I mutant was the sum of the stimulated wild type and the nonstimulated T57I mutant, indicating that T57I did not appreciably compete for YihI (arrows). The E271K mutant was partially stimulated by YihI, and the mixture displayed the sum of both stimulated rates.

YihI stimulation is potassium sensitive.

To directly evaluate the influence of YihI on the Der mutants, we purified YihI so we could monitor its stimulatory effect in GTPase assays. Consistent with previous reports, Der's basal GTPase activity was stimulated by potassium (24, 39). The rate also increased with added KPO4 and did not increase with additional NaCl, so the stimulation was from the potassium ion as has been reported (data not shown) (24, 39). Surprisingly, we discovered that the stimulatory activity of YihI was inversely proportional to potassium stimulation. Without added KCl, YihI increased the weak GTPase rate of wild-type Der approximately 3-fold. At concentrations of potassium greater than ∼250 mM, the stimulation by YihI was lost. Curiously, YihI suppressed the additional stimulation observed avast setup fatal error >250 mM potassium (Fig. 5B; see also the supplemental material). Therefore, YihI stimulation is sensitive to potassium concentration. Viewed another way, YihI helps Der function at lower potassium levels.

YihI fails to bind to or restore the GTPase activity of the Der mutants.

Using wild-type Der, we sought to establish an affinity between these factors under our standard assay conditions (100 mM KCl) by monitoring the increase in Der's GTPase activity as a function of YihI concentration, critical error dea1 l9. Consistent with the previous report of YihI activity under similar conditions (43), we observed a ∼50% increase in Der's GTPase when nearly saturated with YihI (Fig. 5C). We were able to derive a Kd between YihI and Der of 2.6 ± 0.6 μM (see the supplemental material). Thus, the affinity between these factors is moderate and consistent with YihI playing a dynamic regulatory role (43).

Next, we evaluated the ability of YihI to stimulate the L9-dependent Der mutants. We did not detect activation of the T57I mutant at our highest tested concentrations (2 μM Der and 20 μM YihI). The E271K mutant could be stimulated, but only at very high protein concentrations (>2 μM for the E271K mutant and 20 μM for YihI [Fig, critical error dea1 l9. 5C]). Moreover, although the E271K mutation did not reduce GTP binding, it responded similarly to YihI as mutants that have GTP binding site alterations (S16A and S216A) (23, 43). Thus, YihI appears to aid in the turnover of the enzyme but cannot restore critical error dea1 l9 GTPase activities of the mutants despite the fact it partially complements the in vivo phenotypes.

In previous work, it was shown that YihI does not require G domain 1 for binding (43). We were interested in establishing whether YihI could bind to T57I because this protein has a wild-type G domain 2 and KH domain. Therefore, we performed an in-solution competition experiment between the wild type and the T57I mutant for access to YihI. Using our Der-YihI affinity data as a guide (see the supplemental material), we established a condition where the wild-type enzyme was ∼50% occupied by YihI (0.5 μM Der and 2.6 μM YihI). Under this condition, small changes in the available YihI would manifest observable changes in the overall GTPase hydrolysis rate. Because the T57I protein is nearly inactive, if this mutant is capable of binding to YihI to any appreciable extent, an excess of the T57I mutant over the wild type in the mixture should reduce the observed stimulation. We did not observe a reduction in the GTPase stimulation of 0.5 μM wild-type protein using a 5 μM concentration of the T57I mutant as a competitor, and the observed rate was the sum of the stimulated wild-type plus the basal T57I mutant rates (Fig. 5D). Therefore, the T57I mutant had no detectable affinity for YihI. For comparison, adding excess E271K mutant to a reaction mixture containing wild-type Der and YihI caused an increase in GTPase activity that was consistent with both enzymes being stimulated simultaneously. Because Der can function with T57 mutated (when second-site suppressed), these results emphasize a new importance of this highly conserved residue aside from routine GTP hydrolysis.

Purified L9 does not influence Der's GTPase activity.

When purified L9 was added to assays containing wild-type or mutant Der, there was no change in the GTPase rates. Moreover, L9 did not influence Der that was under YihI stimulation (see the supplemental material). This finding suggests that L9's complementation activity in the der mutants may be indirect. Alternatively, the purified L9 may not be active or appropriately presented to Der (technical limitations chronoforms parse error syntax error, unexpected $end us from testing ribosomes with and without L9 at sufficiently high concentrations for these assays).

L9 suppresses an elongated cell morphology caused by derT57I.

The synthetic-lethality analyses revealed that the small-colony phenotype arises when the L9 support plasmid is lost (Fig. 2A). On the surface, this observation could be interpreted in two ways: either L9 accelerated the growth rate of all der mutant cells in a colony (synthetic sickness) or there was a high mortality rate in the der mutants and L9 improves survivability (true synthetic lethality). L9 had the greatest influence on the phenotype of cells with the derT57I allele, so we focused on this mutant for viability and morphology studies.

We were unable to grow homogeneous cultures of the highly compromised ΔrplI derT57I mutant because of the high frequency of second-site suppression (Fig. 6A, left plate). We devised a solution to this problem by employing a targeted protein degradation system to rapidly deplete L9 protein from derT57I cells at a convenient time (29). By allowing L9 to suppress the derT57I allele during culturing, we were able to grow cultures of sufficient size for biochemical analyses. Thinstation no valid session type error this experiment, we modified the derT57I strain in three ways. First, we introduced a functional allele of rplI that encoded a degradation tag on the C terminus of L9 that is recognized by the processive ClpXP protease (rplI-deg); second, we deleted the chromosomal clpX; and third, we introduced a controllable ClpXP expression plasmid. A control strain had a tag on L9 that is not recognized by ClpX (rplI-cont).

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Fig 6

Conditional L9 degradation reveals an unsuppressed derT57I phenotype, critical error dea1 l9. A ΔclpX derT57I strain supported with either L9-cont or L9-deg was transformed with a controllable ClpXP expression plasmid and maintained under noninducing conditions to reduce the accumulation of second-site suppressors. (A) Providing L9-deg to the derT57I strain greatly reduced the accumulation of second-site suppressors. On the left is a representative plate showing the presence of suppressed mutant contaminants when the ΔrplI derT57I strain was grown as an overnight culture without L9 support. On the right is a plate of rplI-deg derT57I ΔclpX cells containing pClpXP that were grown from an overnight culture to late exponential phase in glucose medium (ClpXP off) and then plated on arabinose to induce ClpXP and degrade L9-deg. All colonies were small and reminiscent of freshly isolated, unsuppressed ΔrplIderT57I strains. (B) Cultures of L9-cont and L9-deg were grown to exponential phase and then either treated with glucose (to repress ClpXP expression [circles and triangles]) or induced with arabinose (to express ClpXP [crosses critical error dea1 49 diamonds]). As each fast-growing culture neared the end of exponential phase, aliquots of each were diluted 10-fold into fresh medium to allow extended outgrowth. Separate aliquots were removed for Western analysis of the tagged L9 (top). L9-cont was stable and L9-deg was reduced to very low levels by the first sampling. The growth rate of the culture undergoing L9 degradation was reduced by 36% during the last outgrowth. (C) DIC micrographs of derT57I strains grown with L9 (L9-cont, pClpXP induced) or without L9 (L9-deg, pClpXP induced). Degradation of L9 caused the cells to become elongated. (D) The lengths of 100 cells from each of four different cultures grown with pClpXP induced for three outgrowths were measured from several micrographs and plotted along with their averages (long lines) and standard deviations. Average lengths (μm): L9-cont, der+, 2.53; L9-deg, der+, 2.53; L9-cont, derT57I, 2.84; L9-deg, derT57I, 4.65.

We prepared a dilution of an rplI-deg derT57I culture grown with the proteolysis system off and plated it under conditions with the proteolysis system on. All of the colonies were small and reminiscent of freshly isolated derT57I strains (Fig. 6A, right plate). Thus, the L9-deg protein was capable of suppressing the slow-growth phenotype sufficiently to allow culturing without the accumulation of fast-growing suppressors.

In an rplI-deg derT57I culture, inducing expression of ClpXP caused a rapid depletion of L9-deg (Fig. 6B). Keeping in mind that L9 was still being expressed at high levels as a ribosomal protein, this result indicates that the degradation system was capable of overcoming the L9 synthesis rate and substantially reducing the half-life of the target protein. We noted that extended induction of ClpXP also caused a slight reduction in the L9-cont levels as well, indicating that the protease exhibited partial activity for this tag. This finding also suggests that L9 protein levels may not be autoregulated.

The thorough depletion of L9 occurred by ∼30 min, but we did not observe a pronounced reduction in growth rate until ∼4 subsequent mass doublings had occurred (Fig. 6B). This result is important because it demonstrates that ribosomes (or other important factors) synthesized in the presence of L9 and DerT57I are functional when L9 is removed. Thus, DerT57I likely functions in a difficult biogenesis step that, once overcome, no longer requires the support of L9. In addition, this experiment formally establishes that the protein product of rplI (and not its mRNA) is responsible for the suppression of this der allele.

In a separate set of experiments, we determined that the degradation of L9 in derT57I caused a loss in plating efficiency to ∼40% that of controls (see the supplemental material). We initially attributed this observation to a loss in cell viability. However, microscopic analyses revealed that culturing of the L9-depleted derT57I cells invoked an aberrant, elongated cell morphology (Fig. 6C). The average cell length of L9+derT57I cells was similar to that of L9+der+ cells (2.84 versus 2.53 μm, respectively). In contrast, critical error dea1 l9, when L9 was depleted from the derT57I, the average cell length increased to 4.65 μm, with a high variance, and the distribution of lengths formed clusters, with some cells being longer than 30 μm (Fig. 6D). Thus, the reduction in plating efficiency was likely caused by a reduction in cell division and not from growth inhibition per se. Overall, L9 appears to suppress a cell division defect caused by a crippled Der.


We identified mutations in the highly conserved and essential GTPase Der that cause a dependence on ribosomal protein L9 for improved fitness. The T57I mutant was independently recovered and mapped twice during our screen, probably as daughters of the original mutant that separated during recovery. This mutant displayed a pronounced phenotype and illustrates a balance between the inactivation of an essential enzyme and the ability to identify potential candidates during the visual screening of critical error dea1 l9 library. In G domain 1, T57 is located within the G3 motif, critical error dea1 l9, which connects to the switch II region of this GTPase. Threonine is found at this position in nearly all Der orthologs and could play an important role in the function of the invariant flanking motif residues.

G domain 1 is reported to be responsible for the majority of Der's GTPase activity (23, 39), so breaking the basic catalytic mechanism may explain the highly defective nature of this mutant. However, this GTPase domain is thought to undergo a dramatic reorganization during the GTP hydrolysis cycle. Structures of the Der ortholog from Bacillus subtilis show the G domain rotated such that the T57 location is positioned far away from the KH domain (39). In contrast, in the Thermotoga maritima structure, T57 sits at a well-packed interface between G domain 1 and the RNA-binding KH domain (37). Therefore, the T57I mutation may interfere with the ability of the domain to properly interact with the KH domain. The numerous second-site suppressors of T57I also support an architectural role for this residue, because if it is required for GTP hydrolysis, only revertants should have functioned well.

The E271K mutation sits in the switch II motif of G domain 2 (38). Although there is generally a high variability in switch domains of GTPases (44–46), E271 appears invariant among all Der proteins. Switch motifs in GTPases are thought to couple the energy of GTP hydrolysis to the movement of the switches and allow the enzymes to do mechanical work (44). In the crystal structures of Der, E271 is not in direct contact with residues of the P-loop that gates the GTP hydrolysis site. Nonetheless, this residue is situated at a location that docks this switch against Der in the GTP-bound state. Envisioning a tensioned spring that gets released by GTP hydrolysis during the cycling of the enzyme, a mutation at this location could prevent the formation of a stable, high-energy state of the switch. Our observation that the affinity for GTP of the E271K mutant was comparable to that of the wild type also supports the idea that GTP binding was not inhibited but that the cycling of the enzyme through high- and low-energy states was compromised. Perhaps even more compelling is the observation that the GTPase activity of G domain 1, which is reported to possess the majority of the observed GTPase activity (37), was also substantially inhibited by this mutation.

We observed partial complementation of the phenotypes of each mutant by overexpressing YihI. This factor was identified as an interacting partner of Der that stimulated Der's GTPase rate by both lowering the Km and increasing the Vmax (43). Because YihI was not observed to stimulate GDP release, it was designated a GAP-like factor (43). Unlike canonical GAPs, YihI is reported to stimulate Der only marginally, and our stimulation data were consistent with this conclusion. One interpretation of these findings is that Der is not likely to be a signaling GTPase, critical error dea1 l9, so raising its basal GTPase rate several orders of magnitude, as traditional GAPs do, would not be warranted (47). Alternatively, YihI may not perform a bona fide GAP function by contributing to catalysis and could stimulate the GTPase activity by stabilizing a catalytically active conformation, an idea put forth by its discoverers (43).

We did not observe der mutant complementation by YihI that was better than that provided by L9, suggesting that the growth defects caused by the der mutant persisted in the presence of excess YihI. Moreover, YihI is not universally conserved in bacteria, and it is nonessential (43), critical error dea1 l9, yet overexpression is toxic (suggesting that it can shield Der from important targets). Der has joined a growing list of GTPases that require high potassium levels for optimal activity (24, 39). We discovered that YihI had no stimulatory activity when potassium was present at high levels. Interestingly, the potassium critical error dea1 l9 in E. coli and Klebsiella pneumoniae (in which YihI is present) is reported to fluctuate between ∼100 and ∼250 mM, whereas those of B. subtilis (in which YihI is absent) are maintained at ∼400 mM (39, 48–50). Perhaps YihI helps load Der with potassium or helps Der function when intracellular potassium levels are low.

The goal of this project was to decipher why L9 is conserved in nature, so what is L9's role in Der physiology? Part of the conundrum stems from the fact that we do not know what Der specifically does. Der depletion for extended periods causes the accumulation of unstable and/or incomplete large subunits and defects in both 16S and 23S rRNA processing, which others have suggested points to a role in ribosome biogenesis (23–26, 51). Cells with deficient rRNA folding factors commonly display cold sensitivities (46, 52). We tested for cold critical error dea1 l9 in our der mutants and observed none (data not shown). In addition, another group reported that overexpression of the stringent response factor RelA suppressed the growth defects caused by Der with mutations in either GTPase site, but not a der null mutation (53). A conclusion from that project was that the overexpressed RelA increased (p)ppGpp pools, restricted rRNA synthesis, critical error dea1 l9, and restored balance to the assembly process, critical error dea1 l9. We tested for the ability of overexpressed RelA to suppress the L9-dependent Der mutants, and despite imparting a growth restriction in all strains, we observed no relative fitness increases in the mutants (data not shown). Thus, the T57I and E271K mutants are distinct from Der variants with defective GTPase centers in this regard.

An interesting feature of the large ribosomal subunits recovered from Der-depleted E. coli is that they are sensitive to reduced magnesium levels, suggesting that they have not been assembled correctly (25, 26). The track terrorizer tf2, when these destabilized subunits were evaluated for protein content, L9 was among the few proteins reported to fall off (25, 26). This finding suggests that the binding site of L9 may be compromised when Der activity is reduced. In line with this notion, our studies indicate that the N-terminal domain alone is able to complement derT57I as well as the full-length protein. Additionally, the hop-1 mutation in L9 is in a conserved patch on the C domain, and this variant complements our Der mutants as well as the wild type (data not shown). Considering that there are extensive contacts between the N domain and the 23S RNA, it seems that this portion of L9 may help stabilize the large subunit when Der activity is limiting.

The physiological functions of Der and L9 remain a mystery. An additional approach we took to interrogate the role of L9 in the Der mutants was to critical error dea1 l9 translation bypassing in the mutants using established reporters. Aside from the complication of second-site suppression, we discovered a curious phenomenon that led us to abandon that approach (see the supplemental material).

We used a targeted degradation system to get around a thorny genetic problem and to preliminarily interrogate the physiology of Der mutants as they lose the support of L9. We were pleased to find that the degradation system could deplete L9 so well considering that it is a highly expressed protein that is tightly associated with the ribosome. We plan to use our degradation system to evaluate the integrity of ribosomes built with a defective Der critical error dea1 l9 L9 depletion both in vivo and in vitro. In the preliminary investigation reported here, we revealed an elongation phenotype when the derT57I strain lost the support of L9. We critical error dea1 l9 observed elongated cells in unsupported cultures of derE271K organisms (data not shown). We interpret these results as a problem with cell division caused by a defective Der and not necessarily a problem with biomass accumulation. Thus, der mutants were likely recovered in our screen because the loss of L9 promotes sql error code 17001 retention of the reporter plasmid by reducing cell division. These findings raise interesting new questions about the roles of L9 and Der in ribosome assembly and in maintaining bacterial physiologies.


This work was supported by departmental funds from the Burnett School of Biomedical Sciences, UCF College of Medicine.

We thank Ana C. Carr, William T. Self, Matthew P, critical error dea1 l9. Wood, Alexander M. Cole, Kenneth Teter, and Jingdong Ye, UCF, for their assistance and advice.


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The Review of Economic Studies