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The MiaA tRNA Modification Enzyme Is Necessary for Robust RpoS Expression in Escherichia coli

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The stationary phase/general stress response sigma factor RpoS (σS) is necessary for adaptation and restoration of homeostasis in stationary phase. As a physiological consequence, its levels are tightly regulated at least at two levels. Multiple small regulatory RNA molecules modulate its translation, in a manner that is dependent on the RNA chaperone Hfq and the rpoS 5′ untranslated region. ClpXP and the RssB adaptor protein degrade RpoS, unless it is protected by an anti-adaptor. We here find that, in addition to these posttranscriptional levels of regulation, tRNA modification also affects the steady-state levels of RpoS. We screened mutants of several RNA modification enzymes for an effect on RpoS expression and identified the miaA gene, encoding a tRNA isopentenyltransferase, as necessary for full expression of both an rpoS750-lacZ translational fusion and the RpoS protein. This effect is independent of rpoS, the regulatory RNAs, and RpoS degradation. RpoD steady-state levels were not significantly different in the absence of MiaA, suggesting that this is an RpoS-specific effect. The rpoS coding sequence is significantly enriched for leu codons that use MiaA-modified tRNAs, compared to rpoD and many other genes. Dependence on MiaA may therefore provide yet another way for RpoS levels to respond to growth conditions.
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Thompson and Gottesman
The MiaA tRNA modification enzyme is necessary for robust RpoS expression in
Escherichia coli
Karl M. Thompson
1, #
and Susan Gottesman
2
1
Department of Microbiology
College of Medicine
Howard University
520 W Street, NW
Suite 3010
Washington, DC 20059 

2
Biochemical Genetics Section 
Laboratory of Molecular Biology 
Center for Cancer Research 
National Cancer Institute 
National Institutes of Health 
9000 Rockville Pike 
Building 37, Room 5132 
Bethesda, MD 20892 

#Corresponding Author: Karl M Thompson, Ph.D. (karl.thompson@Howard.edu) 
Running Title: MiaA Role in RpoS Expression 

JB Accepts, published online ahead of print on 2 December 2013
J. Bacteriol. doi:10.1128/JB.01013-13
Copyright © 2013, American Society for Microbiology. All Rights Reserved.
Thompson and Gottesman
ABSTRACT 
The stationary phase / general stress response sigma factor RpoS (
S
) is 
necessary for adaptation and restoration of homeostasis in stationary phase. As a 
physiological consequence, its levels are tightly regulated at least at two levels. Multiple 
small regulatory RNA molecules modulate its translation, in a manner that is dependent 
on the RNA chaperone Hfq and the rpoS 5’ UTR. ClpXP and the RssB adaptor protein 
degrade RpoS, unless it is protected by an anti-adaptor. Here we find that, in addition 
to these post-transcriptional levels of regulation, tRNA modification also affects the 
steady-state levels of RpoS. We screened mutants of several RNA modification 
enzymes for an effect on RpoS expression and identified the miaA gene, encoding a 
tRNA isopentenyltransferase, as necessary for full expression of both an rpoS750-lacZ 
translational fusion and the RpoS protein. This effect is independent of rpoS, the 
regulatory RNAs, and RpoS degradation. RpoD steady-state levels were not 
significantly different in the absence of MiaA, suggesting that this is an RpoS-specific 
effect. The rpoS coding sequence is significantly enriched for leu codons that use 
MiaA-modified tRNAs, compared to rpoD and many other genes. Dependence on MiaA 
may therefore provide yet another way for RpoS levels to respond to growth conditions.
Thompson and Gottesman
INTRODUCTION 
RpoS (
S
) is the stationary phase / general stress response sigma factor 
encoded within the genome of Escherichia coli and other gram-negative enteric bacteria 
(1, 2). RpoS is necessary for cellular adaptation to nutrient deprivation and to the 
presence of toxic metabolites, characteristic of stationary phase cultures. RpoS has a 
vast regulon that facilitates maintenance of cellular homeostasis upon exposure to 
stationary phase conditions (3). RpoS expression and steady-state levels are regulated 
at the transcriptional and post-transcriptional. At the post-transcriptional level, RpoS is 
regulated through both the modulation of translation and protein stability. Hfq, originally 
identified as a host factor for bacteriophage Qβ replication, acts as a chaperone for the 
small regulatory RNAs that interact with the RpoS 5’ UTR (4-8). Three Hfq-dependent 
small regulatory RNAs, DsrA, RprA, and ArcZ, directly stimulate RpoS translation (9-14) 
while OxyS RNA negatively regulates RpoS translation through a mechanism that is not 
completely clear, which likely includes Hfq competition (15-17). In addition to RNA-
mediated regulation of RpoS, the ATP-dependent protease ClpXP, in concert with the 
adaptor protein RssB, degrades RpoS during logarithmic growth (18, 19). Three anti-
adaptor proteins, IraP, IraM, and IraD, stabilize steady-state levels of RpoS protein by 
inhibiting RssB interaction with RpoS, and therefore prevent degradation by ClpXP (20-
22). The tightly controlled steady-state levels of RpoS make it an excellent target to use 
in a screen for novel mechanisms of regulatory control. 
Translational fidelity is necessary for efficient gene expression, and we can 
imagine that high fidelity of translation may be particularly important for expression of 
some genes. The translational machinery contains many components that facilitate 
Thompson and Gottesman
translational fidelity (23). One of these components is modified nucleotides within 
transfer RNAs (tRNAs), specifically those proximal to the anticodon stem-loop (ASL) at 
nucleotide position 37 and at the nucleotide wobble position 34 (24, 25). Modified 
nucleotides differ from standard nucleotides in their structure and composition (26). 
Modified nucleotides proximal to the tRNA anticodon modulate translational fidelity by 
influencing the efficiency of pairing between short regions of complementary bases 
between the mRNA codon and tRNA anticodon (23). These modified nucleotides 
include isomers of normal nucleotides, such as the most abundant modified nucleotide 
pseudouridine, and chemical groups added to existing nucleotides (27, 28). 
Undermodified tRNAs contribute to translational errors and +1 translational 
frameshifting (26, 29-32). Recent reports suggest that the Trm9-catalyzed mcm
5
U34 
(wobble position) tRNA modification may act in a regulatory manner in eukaryotic cells, 
influencing eukaryotic cell cycle progression in response to DNA damage and oxidative 
stress, stimulating the eukaryotic heat shock and unfolded protein responses, and 
perturbing cellular signaling (33-37). Given these observations, it is likely that tRNA 
modifications may play a regulatory role in prokaryotic physiology as well. There is 
relatively little information about the regulatory role that tRNA modifications play in 
prokaryotic cellular physiology. If tRNA modifications have a global role in prokaryotes, 
it is possible that this regulatory role could be effects on global transcriptional regulators 
such as RpoS. While RpoS translation is regulated by SsrA (tmRNA), potentially 
through the limitation of ribosome stalling (38), little is currently known about the role 
that tRNA modification plays in the regulation of RpoS expression or other alternative 
sigma factors in prokaryotic cells. 
Thompson and Gottesman
We screened several tRNA modification mutants, including various 
pseudouridine synthases and the MiaA tRNA prenyl transferase, in order to determine if 
translational fidelity plays a role in the regulatory control of RpoS expression. The miaA 
mutation was the only tested mutation found to affect RpoS expression. 
MiaA catalyzes the first of a two-step tRNA modification process in E. coli and 
Salmonella (39-41). MiaA, along with MiaB, catalyzes the addition of the 2-methylthio-
N6-( 2-isopentenyl), or ms
2
i
6
A, modification to Adenine 37 of tRNAs that recognizes 
codons beginning with uridine (41). The miaA gene is in a complex operon upstream of 
the gene for the RNA chaperone Hfq (42, 43). Mutations in the miaA gene have 
pleiotropic phenotypes (44, 45). Here we show that MiaA is necessary for full 
expression of RpoS. Furthermore, decreased RpoS expression in miaA::kan mutants is 
not due to polarity on the downstream hfq gene or an effect on the RpoS 5’ untranslated 
region, the site of sRNA action. The MiaA effect on RpoS expression appears to be due 
to a defect in translation of the RpoS reading frame, consistent with a direct requirement 
for MiaA for efficient translation of the rpoS mRNA. 
Thompson and Gottesman
MATERIALS AND METHODS 
Strains and Plasmids. Strains and plasmids are listed in Supplementary Tables 1 and 
2 (Tables S1 and S2), respectively. Mutations in miaA, rssB, clpP, hfq, and RNA 
modification genes such as miaA were transferred into the strain carrying either of two 
rpoS-lacZ translational fusions by bacteriophage P1 transduction, selecting for the 
antibiotic resistance markers inserted into these genes. The first fusion, rpoS750-lacZ, 
contains the native rpoS promoters and 5’ UTR; in the second, P
BAD
-rpoS990-lacZ, the 
native promoters have been replaced with P
BAD
, and the 5’ UTR has been deleted. For 
cloning reactions, plasmids were transformed into chemically competent cells using 
heat shock transformation at 42
o
C for 30 seconds. For complementation reactions, 
plasmids were transformed into mutant strains using the TSS transformation method 
(46). 
Construction of pKMT1 (pBAD-miaA) and pKMT2 (pBAD-hfq).
- Plasmid pBAD-miaA 
was constructed by ligation of purified pBAD24 and miaA PCR restriction enzyme 
digests. Briefly, Plasmid pBAD24 was isolated using standard plasmid isolation 
procedures. The miaA gene was amplified via PCR using E. coli MG1655 genomic 
DNA and primers KT01 and KT02 (Table S3). Both purified pBAD24 and the miaA PCR 
product were digested with restriction enzymes EcoRI and PstI. The pBAD24 and miaA 
PCR product digests were ligated using Bacteriophage T4 DNA Ligase. Plasmid pBAD-
hfq was constructed in essentially the same way as plasmid pBAD-miaA, using instead 
primers KT03 and KT04 (Table S3) for the PCR amplification. Each plasmid was 
confirmed by DNA sequencing. 
Thompson and Gottesman
Growth Conditions and Media. Luria Bertani (LB) Lennox liquid media (KD Medical) 
was used for the growth of all liquid cultures. Liquid cultures for β-galactosidase assays 
and / or Western Blots were grown in 125 mL or 250 mL polystyrene Erlenmeyer flasks 
(Corning) at 37
o
C in a shaking water bath. All genetic screens were performed using 
MacConkey-Lactose Agar plates with appropriate antibiotics as needed at 37
o
C. For 
the experiment in Fig. 3A, cultures were grown overnight at 37
o
C in LB Lennox 
supplemented with 0.2% glucose, diluted 1:1000 in LB Lennox supplemented with 0.2% 
glucose and grown at 37
o
C in a shaking water bath until OD600 1.0. 100 uL aliquots 
were taken from the cultures before and after shifting harvested cells to LB Lennox 
supplemented with 0.2% arabinose. Samples for Miller Assays were taken at 5 minute 
intervals after shifting harvested cells to LB Lennox supplemented with 0.2% arabinose. 
-galactosidase assays. -galactosidase assays were carried out in 96-well plates as 
previously described (18). β-galactosidase units are defined as the slope of OD
420

reading divided by OD
600
and are approximately 25-fold lower than Miller Units. Miller 
assays were also performed using a modification of a previously described method (47). 
Briefly, 100 µL aliquots of cell cultures were added to 900 µL of Z-buffer containing 
chloroform and 0.1% SDS. Samples were incubated at 28
o
C until the solution turned 
yellow, after which 500 µL of 1mM Sodium Carbonate was added to each sample. The 
time required for a color change was recorded and the OD420 of each sample was 
measured after centrifugation. Miller Units were calculated as previously defined. Each 
sample was assayed in triplicate for each individual experiment and averages were taken 
as a representative sample for each experiment. The data presented are averages of at 
Thompson and Gottesman
least three independent replicates and error bars represent the standard error of the mean 
(SEM). 
Western Blots. Western Blots were carried out as previously described (18). Briefly, 
100-1000 µL aliquots of cultures were taken at approximately the same OD
600
as 
samples taken for β-galactosidase assays for stationary phase samples and total 
proteins were precipitated by the addition of TriChloroAcetic Acid (TCA) to a final 
concentration of 10% (v/v). These culture aliquots were incubated on ice for 10 minutes 
and total proteins harvested using centrifugation; TCA was removed by rinsing the 
protein pellet with 80% Acetone twice. Cell pellets were mixed with 250 µL of 1X SDS 
Sample Buffer, boiled for 5 minutes, and equal volumes of cell culture were loaded onto 
10% Bis-Tris Novex Gels (Invitrogen – Life Technologies). Total proteins were electro-
transferred onto nitrocellulose membranes and probed using RpoS anti-sera, Goat Anti-
Rabbit Secondary antibody, and using the ECL Kit (Amersham Biosciences – GE 
Healthcare Lifesciences) for detection. 

Thompson and Gottesman
RESULTS 
Mutation of the tRNA modification gene miaA perturbs rpoS-lacZ expression. 
To determine if modified nucleotides within the translational machinery affect 
RpoS expression, we screened several mutants defective in RNA modification activity 
using an rpoS-lacZ translational fusion. We obtained kanamycin-linked deletions of 
several pseudouridine synthase genes, as well as an insertion in miaA, encoding a 
tRNA isopentenyltransferase. These mutations were transduced into a clean genetic 
background strain carrying an rpoS750-lacZ translational fusion using Bacteriophage 
P1. We then tested the phenotype of these transductants on MacConkey-Lactose 
plates (Table 1). The miaA::kan mutation was the only mutation out of those tested that 
resulted in a Lac
-
phenotype, suggesting that MiaA plays a role in the expression of 
RpoS (Table 1, Fig. S1). 

The miaA::kan mutation results in decreased RpoS expression. 
In order to confirm the miaA insertion phenotype seen in the genetic screen, we 
examined RpoS steady-state levels both by β-galactosidase assays of the RpoS-LacZ 
fusion protein and Western blots of the endogenous protein, comparing the wild type 
and miaA strains (Fig. 1). The miaA strain had a defect in β-galactosidase activity (2-3 
fold) throughout the growth of the cultures; this was most notable upon entry into 
stationary phase (OD600 1.5-2.5) (Fig. 1A). Western Blot analysis also showed a 2-3-
fold decrease in both RpoS and RpoS-LacZ protein steady-state levels in the miaA 
strains vs. wild type cultures (Fig. 1B). These results are consistent with the Lac
-

Thompson and Gottesman 
phenotype of miaA mutants in the rpoS750-lacZ fusion strain seen on MacConkey-
Lactose plates (Fig. S1). 

Loss-of-function of MiaA rather than polarity on hfq is responsible for decreased 
RpoS 
The miaA gene is in a complex operon with multiple promoters (Fig. 2A). The miaA 
gene is located directly upstream of the hfq gene, which acts as an RNA chaperone for 
many small regulatory RNAs (sRNAs) (Fig. 2A) (42). One known role of three of these 
sRNAs is to stimulate the translation of rpoS mRNA; hfq mutants are known to have 
decreased steady-state levels of RpoS (6, 48). Therefore, it seemed possible that the 
kan
R
insertion in miaA might decrease RpoS by a polar effect on the hfq gene, reducing 
or eliminating Hfq expression. In fact, there was a clear 2-fold decrease in steady-state 
Hfq levels in a miaA::kan rpoS750-lacZ translational fusion strain compared to a miaA
+

strain (Fig. 2B). 
To distinguish between a direct requirement for MiaA for optimal RpoS synthesis 
and an indirect effect via the polarity on hfq, we measured β-galactosidase activity of 
wild type and miaA rpoS750-lacZ strains transformed with a control vector, pBAD24, 
and either plasmid pBAD-miaA or pBAD-hfq, both with and without arabinose. The 
activity of the miaA::kan rpoS750-lacZ translational fusion strain was low, as previously 
seen, and was restored in the presence of the pBAD-miaA plasmid, in the absence of 
arabinose (Fig. 2C, right panel), suggesting that leaky expression of the miaA gene from 
the P
BAD
promoter is occurring and that minimal amounts of the MiaA protein are 
sufficient for complementation. No such complementation by the pBAD-hfq plasmid 
Thompson and Gottesman 
was seen. These results suggest that while Hfq levels may be decreased in the 
miaA::kan host, it is loss of MiaA and not decreased levels of Hfq that led to decreased 
levels of RpoS seen here. 
For reasons that are not clear, in the presence of arabinose, MiaA expression 
from the pBAD plasmid did not fully restore RpoS expression in the miaA mutant. 
Possibly excess MiaA activity may interfere with RpoS expression, particularly when hfq 
is limiting as seen in Fig. 2B. The pBAD-hfq plasmid also increased the activity of the 
miaA::kan rpoS750-lacZ translational fusion strain in the presence of arabinose. Since 
Hfq is necessary for the activity of three small regulatory RNAs that stimulate 
expression of RpoS (49), we believe that this result suggests that Hfq is limiting for 
sRNA-dependent translation in the miaA::kan strain; high amounts and/or activity of 
these small regulatory RNAs can compensate for the lack of efficient translation in the 
absence of MiaA. Overall, however, the complementation of the miaA::kan mutant by 
pBAD-miaA strongly suggests that MiaA activity is necessary for efficient translation of 
the RpoS mRNAs, and that the phenotype of a miaA::kan is not simply due to polarity 
on hfq. This is further supported by the studies in the next section, demonstrating that 
the miaA effect is independent of the 5’ UTR, the site of Hfq action. 

The MiaA effect on RpoS expression is independent of the RpoS 5’ UTR. 
The fusion used in the experiments above should report on all levels of RpoS 
regulation, since it carries the native rpoS promoter, the 5’ UTR of rpoS, and enough of 
RpoS protein to be recognized and degraded like the wild type RpoS (38). While MiaA 
is expected to act at the level of translation, it could indirectly affect transcription or 
Thompson and Gottesman 
degradation of RpoS. In addition, even if it is acting at the level of translation, the MiaA 
effects on translation could be direct (efficient translation of the RpoS ORF) or indirect, if 
the modification is necessary for synthesis or function of one or more of the small 
regulatory RNAs that act on the RpoS 5’ UTR. 
In order to distinguish between these possibilities, two experiments were done. 
In the first, a translational fusion of rpoS-lacZ was used that differs from that used in Fig. 
1 in two ways (compare Fig. 3B and Fig. 1C). First, transcription is under the control of 
a P
BAD
promoter, so that effects on rpoS transcription are not seen. Second, the long 
leader, subject to Hfq-dependent sRNA regulation, is missing (38). In this strain, an hfq 
mutant had very little effect on levels of RpoS-lacZ, as expected, while a miaA mutant 
significantly reduced expression (Fig. 3A, top graph). Therefore, the miaA mutant 
cannot be acting via an effect on hfq. We introduced an rssB::tet mutation into this 
strain as well; this will block ClpXP-dependent degradation of the RpoS-lacZ fusion. 
The miaA mutant significantly reduced expression of the fusion in this strain as well 
(Fig. 3A, bottom graph), demonstrating that MiaA is not likely to be affecting the 
degradation pathway. In a second experiment, a similar set of experiments examined 
the steady-state level of RpoS itself (rather than a lacZ fusion), in the absence of a 
leader (expression from a pBAD-RpoS plasmid) and in the absence of degradation 
(expression in a clpP mutant) (Fig. 3C). As for Fig. 3A, a miaA mutation lowered the 
level of RpoS in both wild-type and clpP mutant strains (compare lane 4 to 3 and lane 2 
to 1, respectively). If MiaA acted via the degradation pathway, we would expect 
epistasis of the rssB mutant in Fig. 3A and the clpP mutant in Fig. 3C to the miaA 
Thompson and Gottesman 
mutant. These results rule out MiaA effects on stages other than the synthesis of the 
RpoS ORF. 

RpoS steady-state levels are more sensitive to MiaA modified tRNAs than RpoD 
steady-state levels are. 
RpoS is a specialized sigma factor, and it is possible that under some conditions, 
regulation of its translation would be advantageous to the cell. The presence of MiaA 
modified tRNAs, by affecting the steady-state levels of specific protein targets, could act 
to fine-tune the amounts of these global regulators that respond to various stressors. If 
MiaA modulates the steady-state levels of transcriptional regulators that respond to 
specific stressors, like RpoS, we might expect that the MiaA effect on RpoS would not 
be seen on regulators necessary for housekeeping genes such as the vegetative sigma, 
RpoD (
70
). We isolated total protein from miaA
+
and miaA::kan translational fusion 
strains in the exponential and stationary phase of growth and measured the steady-
state levels of RpoD and RpoS (Fig. 4). In comparison to the 2-3 fold decrease in the 
steady-state levels of RpoS in miaA mutants in exponential and stationary phases of 
growth, there was little to no difference in the steady-state levels of RpoD, suggesting 
that MiaA specifically affects RpoS steady-state levels. 





Thompson and Gottesman 
DISCUSSION 
The Steady-State Levels of RpoS have an additional level of regulatory fine-
tuning. 
RpoS is a global regulator whose steady-state levels are important for the proper 
transition of the cell in and out of stationary phase (50). Consequently, multiple genetic 
switches at both the transcriptional and post-transcriptional levels tightly modulate the 
steady state levels of RpoS at all times (50). Transcriptional regulators, multiple small 
regulatory RNA regulators of translation and mRNA stability, and the ClpXP ATP-
dependent protease together with adaptor and anti-adaptor proteins, all act to precisely 
adjust RpoS steady-state levels in response to changes in environmental conditions 
[reviewed in (50)]. Our results suggest that RpoS steady-state levels are influenced by 
yet another regulatory mechanism, translational fidelity via the presence of MiaA-
catalyzed tRNA modifications. MiaB catalyzes a secondary modification, following the 
MiaA-catalyzed prenyl transfer. We tested miaB mutants on the rpoS750-lacZ fusion 
(Table 1) and observed no difference in the Lac phenotype of the fusion, suggesting 
that the RpoS may not have as stringent a requirement for the MiaB-catalyzed 
modification as it does for the MiaA-catalyzed modification. 
These results, in addition to a previous report demonstrating that SsrA is 
necessary for high levels of RpoS translation, supports the idea that translation of the 
RpoS open reading frame is sensitive to the general state of translation (38). What is 
not yet clear are the physiological or environmental conditions under which under-
modified tRNAs are normally present or would affect RpoS. 
Thompson and Gottesman 
Leu Codons recognizing MiaA-modified tRNAs are preferentially enriched in the 
Open Reading Frames of alternative sigma factors, including RpoS. 
Given the difference in miaA sensitivity of RpoS and RpoD, we compared these 
genes to get insight into differences that might explain the MiaA dependence of RpoS. 
MiaA modifies tRNAs that begin with uridine. This includes the only codon for Trp 
(UGG), and the only two codons for Cys, Tyr and Phe. In addition, four of six codons 
encoding Ser start with U; these represent 60% of the codon usage for Ser in E. coli 
(http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topics/in-vitro-genetics/codon-
usage.html). Finally, two of six codons encoding Leu start with U (UUA and UUG), and 
these represent 22% of the total codon usage for Leu. Overall, RpoS has a modestly 
higher ratio of UXX codons and tandem UXX codons to total UXX codons than RpoD 
(0.11 vs 0.08 and 0.17 vs 0.08, respectively). If, however, there were selection within a 
gene for codons that are or are not dependent upon miaA, without changing the amino 
acids, one might expect to see a shift in the use of codons for Leu (an abundant amino 
acid), or Ser. Therefore, we compared the use of UXX codons for Leu in RpoS vs. 
RpoD, as well as in other RNA polymerase components (Table 2). Overall, 28% of the 
Leu codons in RpoS are UUX codons, slightly higher than the 22% predicted from 
analysis of the full genome. For RpoD, UUX codons represented 10% of the overall 
Leu codons, lower than expected. The difference was even more striking in the first 60 
amino acids of each protein, in which RpoD had 0/3 UUX codons, but RpoS had 5/7 
UUX codons. We note that we see the effect of the miaA mutation in the RpoS-lacZ 
fusion, in which only 250 codons of RpoS are present. We extended our analysis to 
look at other components of RNA polymerase and the miaA operon itself (Table 2). The 
Thompson and Gottesman 
core proteins of RNA polymerase generally have a very low frequency of MiaA-modified 
leu codons. In the first 60 amino acids, RpoS has the highest frequency of MiaA-
modified leu codons of the specialized sigma factors, although both RpoE and FecI 
have levels higher than expected (Table 2). Three of the ten proteins encoded by the 
miaA operon have high levels, and these are all involved in translation – tsaE, encoding 
a protein involved in the biosynthesis of the t
6
A tRNA modification of tRNAs reading 
AXX codon (51), miaA itself and hfq. Thus, part of the decrease in Hfq in a miaA 
mutant may reflect loss of MiaA activity for Hfq translation rather than polarity. Our 
findings here suggest that a global analysis of the distribution of use of modified and 
unmodified codons may provide further insight into regulatory consequences of these 
modifications. 
E. coli RpoS is not the only global regulatory protein responsive to the levels of 
MiaA modified tRNAs in the bacterial cell, as two other bacteria have global regulators 
that require MiaA for full expression (52-54). Agrobacterium tumefaciens vir gene 
expression was decreased 2-10 fold in the absence of MiaA and upon acetosyringone 
induction (52). Shigella flexneri VirF steady-state levels were decreased by 10-fold in 
cells lacking MiaA (53, 54). These previous reports, along with our data, suggest that 
undermodified tRNAs may have an aberrant effect on the translation of global 
regulators. We note that virF had an even higher fraction of UUX leu codons (0.64 
overall, 1.0 in first 60 nt) than RpoS. In Streptomyces coelicolor, tRNA


suppresses 
the bld mutant phenotype, characterized by defective mycelium formation (55). The 
codons that read tRNA


should be MiaA-modified. These results suggest that there 
has been evolution of codon use to make some genes and not others highly dependent 
Thompson and Gottesman 
upon MiaA modification of tRNAs. It would be very efficient, from a regulatory 
standpoint, for the open reading frames of global regulators to be more sensitive to 
undermodified tRNAs than the open reading frames of structural proteins or enzymes. 
Defining the growth conditions when MiaA modification may be limiting or 
particularly important for maintaining robust translation will provide insight into how this 
modification is used to adjust cell physiology. There are some clues from past work. In 
Salmonella but not in E. coli, the absence of miaA lowered expression of the leu operon 
at high temperatures (56). In E. coli, mutation or deletion of a rare tRNA capable of 
reading UUG Leu codons, tRNA

or tRNA


encoded by leuX, made cells dependent 
upon miaA for growth at high temperatures and for efficient translation of UUG-enriched 
LacZ, suggesting that the MiaA modification is particularly important to allow tRNA

, or 
tRNA


encoded by leuZ, to recognize UUG codons at elevated temperatures (57). 
The temperature sensitivity phenotypes are consistent with the presence of a heat-
shock promoter upstream of the miaA gene (43, 58). These previous reports, along 
with our data, suggest a possible link between leucine metabolism and the general 
stress response mediated by RpoS. 

The MiaA effect on RpoS steady-state levels may explain its genomic proximity to 
Hfq. 
The miaA gene (42, 43) is in a complex operon with multiple promoters, directly 
upstream of the hfq gene. Furthermore, the expression of hfq is directly influenced by 
the miaA gene, as there are 3 hfq promoters within the miaA open reading frame (42, 
43). Since the classical definition of an operon includes both co-transcription and 
Thompson and Gottesman 
functional similarity, one may expect some related biochemical or physiological function 
between MiaA and Hfq. All of the small regulatory RNAs that regulate RpoS steady-
state levels are Hfq-dependent. In the absence of Hfq, RpoS steady-state levels are 
severely decreased (12). Our studies provide insight into a physiological relationship 
between MiaA and Hfq, by identifying MiaA as a protein that is also necessary for wild-
type RpoS steady-state levels in the cell. 

Acknowledgements: We would like to thank Nancy Gutgsell and James Ofengand (U. 
Miami) for providing RNA modification mutants and Malcolm Winkler (Indiana U.) for 
sending us miaA mutations. We would like to thank Gisela Storz, Kumaran 
Ramamurthi, Nadim Majdalani, Nicolas DeLay, Hyun-Jung Lee, and Valerie deCrecy-
Lagard for providing comments on this manuscript. We would also like to thank Joseph 
Aubee for assistance with experiments done during revision. This research was 
supported in part by the Intramural Research Program of the NIH, National Cancer 
Institute, Center for Cancer Research. KMT was supported in part through startup 
funding from Howard University College of Medicine. 





Thompson and Gottesman 
Figure Legends 
Figure 1. MiaA effect on rpoS750-lacZ translation fusion activity and RpoS 
steady-state levels. A. Wild type and miaA strains were grown overnight at 37
o
C. 
Overnight cultures were diluted 1:1000 into 50 mL of fresh LB in a 250 mL Erlenmeyer 
flask and grown at 37
o
C in a shaking water bath at 250 rpm. Culture aliquots were 
taken throughout growth of the cultures for β-galactosidase activity measurements. B. 
Overnight cultures of wild type (EM1050) and miaA (KMT31) rpoS750-lacZ translational 
fusion strains grown at 37
o
C were diluted 1:1000 into 50 mL of fresh LB in a 250 mL 
Erlenmeyer flask. Cultures were grown to an OD
600
of 1.5 to 2.0 and total protein was 
isolated by 10% TCA precipitation and subjected to Western Blot analysis using 
polyclonal RpoS anti-sera. Volume of total cell lysates was adjusted so that equivalent 
OD
600
units were loaded for the WT and miaA samples. For wild type and mutant 
samples, the three lanes represent 10 µl, 5 µl, and 1µl of sample loaded onto the gel. 
C. rpoS750-lacZ native translational fusion schematic, highlighting fusion components 
such as the RpoS 5’ UTR responsive to small Hfq and several RpoS regulation small 
RNAs as well as the ClpXP degradation signal. 
Figure 2. MiaA effects are independent of Hfq polarity. A. Genetic and 
transcriptional organization of the complex yjeF-tsaE-amiB-mutL-miaA-hfq-hflX-hflK-hflC 
operon. The approximate location of the miaA::kan insertion mutation is highlighted. B. 
Overnight cultures of wild type (EM1050) and miaA rpoS750-lacZ (KMT31) translational 
fusion strains were grown as described in Fig. 1B and subjected to Western Blot 
analysis using Polyclonal Hfq anti-sera pre-adsorbed to crude cell lysates of hfq mutant 
cultures. These were the same samples analyzed in Fig. 1B. C. Cultures of wild type 
Thompson and Gottesman 
rpoS750-lacZ and miaA rpoS750-lacZ strains containing pBAD24 (KMT69; KMT54), 
pBAD-miaA (KMT70; KMT55), and pBAD-hfq (KMT71; KMT56) were grown in 50mL of 
LB (Lennox) liquid media supplemented with 50 µg/mL of ampicillin. These cultures 
were grown at 37
o
C in shaking water baths, without or with 0.002% arabinose to induce 
expression of either miaA or hfq. Cultures were grown to early stationary phase, OD
600

1.5-2.0, and 100 L culture aliquots were taken for -galactosidase activity 
measurements. Each value represents the mean of at least three replicate 
experiments; the error bars represent standard error of the mean (SEM). 
Figure 3. Expression of leaderless RpoS in the absence of MiaA. 
A. Overnight cultures of wild type (CRB316), miaA (KMT582), hfq (KMT581), rssB 
(KMT583) and rssB miaA (KMT584) P
BAD
-rpoS990-lacZ translational fusion strains were 
grown in LB + 0.2% glucose at 37
o
C to an OD
600
of 1.0. Cultures were collected and 
resuspended in LB + 0.2% arabinose and 100 µL aliquots were taken every 5 min for β-
galactosidase assays. Each value represents the mean of at least three replicate 
experiments; the error bars represent standard error of the mean (SEM). B. Schematic 
of the genetic organization of the chromosomal P
BAD
-rpoS990-lacZ translational fusion 
used in A. C. Cultures of a rpoS::Tn10 strain (KMT80), and its isogenic derivatives, 
miaA (KMT83), clpP (KMT75), clpP miaA (KMT99) strains, each containing a pBAD24-
rpoS plasmid were grown at 30
o
C in LB + ampicillin to exponential phase (OD
600
0.5-
0.7). Total protein was isolated by TCA precipitation and subjected to Western Blot 
analysis using rabbit polyclonal anti-sera against RpoS (
S
). The pBAD24-rpoS plasmid 
does not carry any of the 5’ UTR of rpoS. 
Thompson and Gottesman 
Figure 4. Steady-state levels of RpoS and RpoD proteins in the absence of MiaA. 
Overnight cultures of wild type (EM1050) and miaA (KMT31) rpoS750-lacZ translational 
fusion strains grown at 37
o
C were diluted 1:1000 into 50 mL of fresh LB in a 250 mL 
Erlenmeyer flask. Cultures were grown to exponential phase (OD
600
of 0.5-0.7) or early 
stationary phase of growth (OD
600
of 1.5-2.0) and total protein was isolated by 10% TCA 
precipitation and was subjected to Western Blot analysis using Rabbit Polyclonal anti-
sera against RpoD (
70
) and RpoS (
S
). 


Thompson and Gottesman 
Tables 
Table 1. tRNA modifications screened for an effect on RpoS expression. 
Genotype
a
Biochemical activity removed by mutation
Lac
wild type None +
truA tRNA Pseudouridine synthase, anticodon stem-loop specific +
truB tRNA Pseudouridine synthase,55 specific +
rluA Pseudouridine synthase, 23S rRNA and tRNA
phe
specific +
rluC
Pseudouridine synthase, 23S rRNA, positions 955, 2504, and 2580 +
rsuA Pseudouridine synthase, 16S rRNA position 516 +
miaA UXX codon tRNA prenyl transferase at position 37 -
miaB UXX codon
tRNA methylthiolase following prenylation +

a
The mutations were transduced into the wild type rpoS750-lacZ translational fusion strain (EM1050) and 
tested for expression phenotypes on MacConkey-Lactose Plates incubated overnight at 37°C. Strain 
names are given in Table S1. 














Thompson and Gottesman 
Table 2 - Leucine Codon Usage in regulatory genes 
Gene
Full Orf First 60
RNA Polymerase genes Fraction UUX Leu
a
Fraction UUX Leu
RNAP Components
rpoD
0.06 0.00
rpoA
0.10 0.13
rpoB
0.06 0.00
rpoC
0.03 0.40
Specialized Sigma Factors
rpoS
0.29 0.63
rpoN
0.20 0.21
rpoE
0.39 0.40
rpoF / fliA
0.25 0.18
rpoH
0.21 0.30
fecI
0.14 0.43
hfq operon
yjeF
0.27 0.00
tsaE
0.42 0.43
amiB
0.29 0.29
miaA
0.46 0.58
hfq
0.43 0.43
mutL
0.27 0.20
hflK
0.10 0.25
hflC
0.05 0.00
hflX
0.30 0.40
Shigella flexneri virF
0.64 1
Leucine codon use: Expectation: 0.22
b
a. Codon Usage Determination: Protein sequences of interest were obtained from EcoCyc and pasted 
into the codon usage analysis of the Sequence Manipulation Suite 
(
http://www.bioinformatics.org/SMS/). 
b. Codon frequency in E. coli:
http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topics/in- 
vitro-genetics/codon-usage.htm 


Thompson and Gottesman 
REFERENCES 
 Lange R, Hengge-Aronis R.  )dentification of a central regulator of stationaryphase 
gene expression in Escherichia coli Mol Microbiol 5: 
 Tanaka K, Takayanagi Y, Fujita N, Ishihama A, Takahashi H.  (eterogeneity of the 
principal sigma factor in Escherichia coli the rpoS gene product sigma  is a second 
principal sigma factor of RNA polymerase in stationaryphase Escherichia coli Proc Nat 
Acad Sci USA 90: 
 Battesti A, Gottesman S.  Roles of adaptor proteins in regulation of bacterial 
proteolysis Current Opinion Microbiol 16: 
 Kajitani M, Ishihama A.  )dentification and sequence determination of the host factor 
gene for bacteriophage Q beta Nucleic Acids Res 19: 
 Muffler A, Fischer A, Hengge-Aronis R. The RNAbinding protein (F known as a 
host factor for phage Qbeta RNA replication is essential for rpoS translation in Escherichia 
coli Genes Dev 10: 
 Brown L, Elliot T.  Efficient translation of the RpoS sigma factor in Salmonella 
typhimurium requires host factor ) an RNAbinding protein encoded by the hfq gene J 
Bacteriol 178: 
 Cunning C, Brown L, Elliott T.  Promoter substitution and deletion analysis of 
upstream region required for rpoS translational regulation J Bacteriol 180: 
 Soper T, Mandin P, Majdalani N, Gottesman S, Woodson SA.  Positive regulation by 
small RNAs and the role of (fq Proc Nat Acad Sci USA 107:

Brown L, Elliot T.  Mutations that increase expression of the rpoS gene and decrease 
its dependence on hfq function in Salmonella typhimurium J Bacteriol 179: 
Thompson and Gottesman 
 Majdalani N, Cunning C, Sledjeski DD, Elliott T, Gottesman S. DsrA RNA regulates 
translation of RpoS message by an antiantisense mechanism independent of its action as 
an antisilencer of transcription Proc Nat Acad Sci USA 95: 
 Majdalani N, Chen S, Murrow J, St John K, Gottesman S.  Regulation of RpoS by a 
novel small RNA the characterization of RprA Mol Microbiol 39:
 Muffler A, Fischer D, Hengge-Aronis R. The RNAbinding protein (F known as a 
host factor for phage Qbeta RNA replication is essential for rpoS translation in Escherichia 
coli Genes Dev 10: 
 Sledjeski DD, Gupta A, Gottesman S.  The small RNA DsrA is essential for the low 
temperature expression of RpoS during exponential growth in Escherichia coli EMBO J
15: 
 Mandin P, Gottesman S.  )ntegrating anaerobicaerobic sensing and the general 
stress response through the ArcZ small RNA EMBO J 29: 
 Zhang A, Altuvia S, Storz G.  The novel oxyS RNA regulates expression of the sigma s 
subunit of Escherichia coli RNA polymerase Nucleic acids symposium series 36: 
 Zhang A, Altuvia S, Tiwari A, Argaman L, Hengge-Aronis R, Storz G.  The OxyS 
regulatory RNA represses rpoS translation and binds the (fq (F) protein EMBO J 
17: 
 Moon K, Gottesman S.  Competition among (fqbinding small RNAs in Escherichia 
coli Molecular microbiology 82: 

Zhou Y, Gottesman S. Regulation of proteolysis of the stationaryphase sigma 
factor RpoS J of Bacteriol 180:
 Muffler A, Fischer D, Altuvia S, Storz G, Hengge-Aronis R.  The response regulator 
RssB controls stability of the sigmaS subunit of RNA polymerase in Escherichia coli EMBO 
J 15: 
Thompson and Gottesman 
 Tu X, Latifi T, Bougdour A, Gottesman S, Groisman EA.  The PhoPPhoQ two
component system stabilizes the alternative sigma factor RpoS in Salmonella enterica Proc 
Nat Acad Sci USA 103: 
 Bougdour A, Gottesman S.  ppGpp regulation of RpoS degradation via antiadaptor 
protein )raP Proc Nat Acad Sci USA 104: 
 Bougdour A, Cunning C, Baptiste PJ, Elliott T, Gottesman S.  Multiple pathways for 
regulation of sigmaS RpoS stability in Escherichia coli via the action of multiple anti
adaptors Molecular microbiology 68: 
 Bjork GR, Durand JM, Hagervall TG, Leipuviene R, Lundgren HK, Nilsson K, Chen P, 
Qian Q, Urbonavicius J.  Transfer RNA modification influence on translational 
frameshifting and metabolism FEBS letters 452: 
 Gustilo EM, Vendeix FAP, Agris PF.  tRNAs modifications bring order to gene 
expression Current opinion in microbiology 11: 
 Agris PF, Vendeix FAP, D. GW.  tRNAs wobble decoding of the genome  years of 
modification J Mol Biol 366: 
 El Yacoubi B, Bailly M, de Crecy-Lagard V.  Biosynthesis and function of 
posttranscriptional modifications of transfer RNAs Annu Rev Genetics 46: 
 Ofengand J, Malhotra A, Remme J, Gutgsell NS, Del Campo M, Jean-Charles S, Peil L, 
Kaya Y.  Pseudouridines and pseudouridines synthases of the ribosome Cold Spring 
(arb Symp Quant Biol 66: 
 Grosjean H, Benne R.  Modification and editing of RNA American Society for 
Microbiology Press 
 Hagervall TG, Ericson JU, Esberg KB, Li JN, Bjork GR.  Role of tRNA modification in 
translational fidelity Biochimica et biophysica acta 1050: 
Thompson and Gottesman 
 Urbonavicius J, Stahl G, Durand JM, Ben Salem SN, Qian Q, Farabaugh P, Bjork GR. 
 Transfer RNA modifications that alter  frameshifting in general fail to affect  
frameshifting Rna 9: 
 Urbonavicius J, Qian Q, Durand JM, Hagervall TG, Bjork GR.  )mprovement of 
reading frame maintenance is a common function for several tRNA modifications EMBO J 
20: 
 Qian Q, Bjork GR.  Structural alterations far from the anticodon of the tRNAProGGG of 
Salmonella typhimurium induce  frameshifting at the peptidylsite J Mol Biol 273:
 
 Patil A, Dyavaiah M, Joseph F, Rooney JP, Chan CT, Dedon PC, Begley TJ.  
)ncreased tRNA modification and genespecific codon usage regulate cell cycle progression 
during the DNA damage response Cell Cycle 1: 
 Patil A, Chan CT, Dyavaiah M, Rooney JP, Dedon PC, Begley TJ.  Translational 
infidelityinduced protein stress results from a deficiency in Trmcatalyzed tRNA 
modifications RNA Biol 9: 
 Chan CT, Pang YL, Deng W, Babu IR, Dyavaiah M, Begley TJ, Dedon PC.  
Reprogramming of tRNA modifications controls the oxidative stress response by codon
biased translation of proteins Nat Commun 3: 
 Begley TJ, Dyavaiah M, Patil A, Rooney JP, Direnzo D, Young CM, Conklin DS, Zitomer 
RS, Begley TJ.  Trm catalized tRNA modifications link translation to the DNA 
damage response Mol Cell 28: 
 Zinshteyn B, Gilbert WV.  Loss of a conserved tRNA anticodon modification perturbs 
cellular signaling PLOS Genetics 9:e 
 Ranquet C, Gottesman S.  Translational regulation of the
Escherichia coli stress factor 
RpoS a role for SsrA and Lon J Bacteriol 189: 
Thompson and Gottesman 
 Buck M, Ames BN.  A modified nucleotide in tRNA as a possible regulator of 
aerobiosis synthesis of cismethylthioribosylzeatin in the tRNA of Salmonella Cell 
36: 
 Petrullo LA, Elseviers D.  Effect of a methylthioNisopentenyladenosine 
deficiency on peptidyltRNA release in Escherichia coli J Bacteriol 165: 
 Connolly DM, Winkler ME.  Genetic and physiological relationships among the miaA 
gene methylthioNdelta isopentenyladenosine tRNA modification and 
spontaneous mutagenesis in Escherichia coli K Journal of bacteriology 171: 
 Tsui HC, Winkler ME.  Transcriptional patterns of the mutLmiaA superoperon of 
Escherichia coli K suggest a model for posttranscriptional regulation Biochimie 
76: 
 Tsui HC, Feng G, Winkler ME.  Transcription of the mutL repair miaA tRNA 
modification hfq pleiotropic regulator and hflA region protease genes of Escherichia coli K
 from clustered Esigmaspecific promoters during heat shock J Bacteriol 178:
 
 Connolly DM, Winkler ME.  Structure of Escherichia coli K miaA and 
characterization of the mutator phenotype caused by miaA insertion mutations J Bacteriol 
173: 
 Tsui HC, Leung HC, Winkler ME.
 Characterization of broadly pleiotropic phenotypes 
caused by an hfq insertion mutation in Escherichia coli K Mol Microbiol 13: 
 Chung CT, Niemela SL, Miller RH.  Onestep preparation of competent Escherichia 
coli transformation and storage of bacterial cells in the same solutions Proc Nat Acad Sci 
USA 86: 
 Miller JH.  A short course in bacterial genetics Cold Spring (arbor Laboratory Press 
Plainview N Y 
Thompson and Gottesman 
 Muffler A, Fischer A, Hengge-Aronis R. The RNAbinding protein (F) known as a 
host factor for phage Qbeta RNA replication is essential for rpoS translation in Escherichia 
coli Genes Dev 10: 
 Gottesman S.  The small RNA regulators of Escherichia coli roles and mechanisms 
Annu Rev Microbiol 58: 
 Battesti A, Majdalani N, Gottesman S.  The RpoSMediated General Stress Response 
in Escherichia coli Ann Rev of Microbiol 65: 
 Deutsch C, El Yacoubi B, de Crecy-Lagard V, Iwata-Reuyl D.  Biosynthesis of 
threonylcarbamoyl adenosine t
A a universal tRNA nucleoside J Biol Chem 287:
 
 Gray J, Wang J, Gelvin SB.  Mutation of the miaA gene of Agrobacterium tumefaciens 
results in reduced vir gene expression J Bacteriol174: 
 Durand JM, Bjork GR, Kuwae A, Yoshikawa M, Sasakawa C.  The modified 
nucleoside methylthioNisopentenyladenosine in tRNA of Shigella flexneri is required 
for expression of virulence genes J of Bacteriol 179: 
 Durand JM, Dagberg B, Uhlin BE, Bjork GR.  Transfer RNA modification 
temperature and DNA superhelicity have a common target in the regulatory network of the 
virulence of Shigella flexneri the expression of the virF gene Mol Microbiol 35: 
 Pettersson BM, Kirsebom LA.  tRNA accumulation and suppression of the bldA 
phenotype during development in Streptomyces coelicolor Mol Microbiol 79: 
 Blum PH.  Reduced leu operon expression in a miaA mutant of Salmonella 
typhimurium J Bacteriol 170: 
 Nakayashiki T, Inokuchi H.  Novel temperaturesensitive mutants of Escherichia coli 
that are unable to grow in the absence of wild type tRNA
Leu
 J Bacteriol 180: 
Thompson and Gottesman 
 Nonaka G, Blankschien M, Herman C, Gross CA, Rhodius VA.  Regulon and 
promoter analysis of the E. coli heatshock factor sigma reveals a multifaceted cellular 
response to heat stress Genes Dev 20: 

... For example, tRNAs can be covalently modified by >100 different moieties that can influence the charging of tRNAs with amino acids, tRNA stability, codon usage, and reading frame maintenance (1)(2)(3)(4). In Escherichia coli and other organisms, the hypomodification of tRNAs can result in decreased growth rates, altered metabolic requirements, and reduced stress resistance (5)(6)(7)(8). Shifts in the prevalence of specific tRNA modifications are proposed to help optimize cellular responses to stress by affecting translational fidelity and selective protein expression (9)(10)(11). In other words, changing levels of tRNA modifications may control the codon-biased translation of select transcripts, providing a post-transcriptional programmable mechanism that distressed cells can use to facilitate beneficial changes in their proteomes. ...
... Mutations in the miaA locus result in an unmodified A-37 residue, as prenylation is required for methylthiolation by MiaB. In laboratory-adapted K-12 E. coli strains, mutations in miaA impair attenuation of the tryptophan and phenylalanine operons (20,21) and diminish translation of the stationary phase sigma factor RpoS and the small RNA chaperone Hfq (7,22,23). Additionally, mutants lacking miaA are unable to effectively resolve aberrant DNA-protein crosslinks (24) and have somewhat elevated spontaneous mutation frequencies (10,25,26). The ms 2 i 6 A-37 modification is highly conserved in both prokaryotes and eukaryotes, though the specific enzymes that mediate this modification have diverged within evolutionarily distant organisms (8). ...
... In K-12 E. coli, the deletion of miaA results in decreased translation of the alternate Sigma factor RpoS ( S ) and the small RNA chaperone Hfq (7,22,23). Both of these factors are important for the stress resistance and virulence potential of ExPEC (68,87). ...
Article
Full-text available
Post-transcriptional modifications can impact the stability and functionality of many different classes of RNA molecules and are an especially important aspect of tRNA regulation. It is hypothesized that cells can orchestrate rapid responses to changing environmental conditions by adjusting the specific types and levels of tRNA modifications. We uncovered strong evidence in support of this tRNA global regulation hypothesis by examining effects of the well-conserved tRNA modifying enzyme MiaA in extraintestinal pathogenic Escherichia coli (ExPEC), a major cause of urinary tract and bloodstream infections. MiaA mediates the prenylation of adenosine-37 within tRNAs that decode UNN codons, and we found it to be crucial to the fitness and virulence of ExPEC. MiaA levels shifted in response to stress via a post-transcriptional mechanism, resulting in marked changes in the amounts of fully modified MiaA substrates. Both ablation and forced overproduction of MiaA stimulated translational frameshifting and profoundly altered the ExPEC proteome, with variable effects attributable to UNN content, changes in the catalytic activity of MiaA, or availability of metabolic precursors. Cumulatively, these data indicate that balanced input from MiaA is critical for optimizing cellular responses, with MiaA acting much like a rheostat that can be used to realign global protein expression patterns.
... This modification stabilizes codon-anticodon interaction, thereby increasing translational speed and fidelity [7]. In E.coli, MiaA catalyses the transfer of an isopentenyl group from dimethylallylpyrophosphate (DMAPP) to N6 position of adenosine, which is essential for the transition of the bacteria to stationary phase [8]. MiaA is also associated with virulence in pathogenic bacteria like Shigella flexneri, where mutation in the gene affected expression of many virulence genes [9]. ...
... For example, in E.coli stress transcription factor RpoS needs i6A modified tRNAs for its translation as the RpoS mRNA is enriched with LeuUUA codons. So lack of i6A modification impairs the successful transition of E.coli to stationary phase [8,19]. But in Extraintestinal Pathogenic E.coli (ExPEC), miaA is found essential for the gut colonization; miaA mutant strains have limited metabolic flexibility and virulence in comparison with the wild type. ...
Article
tRNA modifications play a significant role in the structural stability as well as translational fidelity in all organisms from bacteria to humans. They also play a major role in bacterial physiology by regulating translation in response to various environmental stresses. Modifications coming at the anticodon-stem loop (ASL) are particularly important as they stabilize codon-anticodon interactions, ensuring accuracy and speed in decoding mRNAs Addition of isopentenyl group (i6A) at A37 position by tRNA isopentenyltransferase (MiaA) is a well conserved modification from bacteria to human. We studied M. tuberculosis MiaA from strain H37Rv and identified the target tRNAs for this modification based on the A36A37A38 motif. i6A modification of target tRNAs tRNALeuCAA, tRNAPheGAA, tRNATrpCCA and tRNASerCGA were further confirmed by isopentenyltransferase assay providing the substrate DMAPP and recombinant MiaA enzyme.
... Among the identified genes from our screening, miaA, prfC, rplI, and rsmG are translational factors that have been previously associated with translational fidelity. MiaA catalyzes the isopentenyladenosine modification at position 37 (i 6 A37) of tRNAs and its deletion decreases UGA readthrough (39); it also plays an important role in the general stress response (40). The prfC gene encodes RF3, which facilitates RF1 and RF2 to terminate translation at stop codons (41). ...
Preprint
Full-text available
Translational quality control is critical for maintaining the accuracy of protein synthesis in all domains of life. Mutations in aminoacyl-tRNA synthetases and the ribosome are known to affect translational fidelity and alter fitness, viability, stress responses, neuron function, and life span. In this study, we used a high-throughput fluorescence-based assay to screen a knock-out library of Escherichia coli and identified 30 nonessential genes that are critical for maintaining the fidelity of stop-codon readthrough. Most of these identified genes have not been shown to affect translational fidelity previously. Intriguingly, we show that several genes controlling metabolism, including cyaA and guaA , unexpectedly enhance stop-codon readthrough. CyaA and GuaA catalyze the synthesis of cyclic adenosine monophosphate (cAMP) and guanosine monophosphate (GMP), respectively. Both CyaA and GuaA increase the expression of ribosomes and tRNAs, allowing aminoacyl-tRNAs to compete with release factors and suppress stop codons. In addition, the effect of guaA deletion on stop-codon readthrough is abolished by deleting prfC , which encodes release factor 3 (RF3). Our results suggest that nucleotide and carbon metabolism is tightly coupled with translational fidelity.
... MiaA (dimethylallyl diphosphate:tRNA transferase) is a tRNA-modifying enzyme reported to contribute toward efficient translation of RpoS and IraP. 90,91 RpoS, the alternate sigma factor, is known to regulate expression of genes operating in the stationary phase and under various stress conditions. 92 On the other hand, IraP is an antiadaptor protein, which stabilizes the levels of RpoS by preventing the RssB-mediated degradation of RpoS. ...
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... MiaB acts downstream of MiaA, which is a tRNA isopentenyltransferase. MiaA uses DMAPP to isopentenylate the N 6 -nitrogen of adenine at base 37 (A37), forming the i 6 A37 modification (39,67), on a subset of tRNAs that read codons beginning with uridine (Phe, Leu, Ser, Tyr, Cys, and Trp) (67,68). Downstream of this, MiaB functions as a tRNA methylthiotransferase (19), catalyzing the methylthiolation of the i 6 A37 base, to generate the FIG 5 Roles of FeS-dependent proteins in the apicoplast. ...
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... MiaA (dimethylallyl diphosphate:tRNA transferase) is a tRNA-modifying enzyme reported to contribute toward efficient translation of RpoS and IraP. 90,91 RpoS, the alternate sigma factor, is known to regulate expression of genes operating in the stationary phase and under various stress conditions. 92 On the other hand, IraP is an antiadaptor protein, which stabilizes the levels of RpoS by preventing the RssB-mediated degradation of RpoS. ...
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... In E. coli, it has been shown that ms 2 i 6 A is important for translation of RpoS, the general stress response alternative sigma factor, which is particularly rich in UUX-Leu codons over CUX-Leu codons [150]. Similarly, S. flexneri virF contains a high proportion of UUX-Leu codons [151]. Although the mechanism by which Q induces virF translation is not known, it is noticeable that putrescine or a combination of methionine and arginine metabolically related to putrescine, restore VirF expression of S. flexneri tgt mutant [152]. ...
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... However, only few studies link cellular adaptation response to stress conditions to dynamic changes of specific tRNAs and provide a molecular basis for the impact of modification levels on gene expression. For instance, MiaB-mediated ms 2 i 6 A37 modification in Escherichia coli tRNA plays a role in stress response by modulating the steady-state level of the stress-induced transcription factor RpoS (33). Moreover, a study on Mycobacterium bovis demonstrated that early hypoxia induces wobble modification cmo 5 U in tRNA Thr (cmo 5 UGU), thus leading to codon-biased translation (34). ...
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Thesis
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