Mutational analysis of bacteriophage T4 AsiA: involvement of N- and C-terminal regions in binding to σ70 of Escherichia coli in vivo

Gene ◽  
2002 ◽  
Vol 295 (1) ◽  
pp. 125-134 ◽  
Author(s):  
Umender K. Sharma ◽  
P.V.K. Praveen ◽  
T.S. Balganesh
Keyword(s):  
Escherichia Coli ◽  
Mutational Analysis ◽  
Bacteriophage T4

scholarly journals Mutational analysis of the mRNA operator for T4 DNA polymerase.

Genetics ◽  
1991 ◽  
Vol 128 (2) ◽  
pp. 203-213 ◽  
Author(s):  
M D Andrake ◽  
J D Karam
Keyword(s):  
Dna Polymerase ◽  
Mutational Analysis ◽  
Bacteriophage T4 ◽  
Spatial Arrangement ◽  
Loop Sequence ◽  
Shine Dalgarno Sequence ◽  
In Vitro Effects ◽  
Rna Hairpin

Abstract Biosynthesis of bacteriophage T4 DNA polymerase is autogenously regulated at the translational level. The enzyme, product of gene 43, represses its own translation by binding to its mRNA 5' to the initiator AUG at a 36-40 nucleotide segment that includes the Shine-Dalgarno sequence and a putative RNA hairpin structure consisting of a 5-base-pair stem and an 8-base loop. We constructed mutations that either disrupted the stem or altered specific loop residues of the hairpin and found that many of these mutations, including single-base changes in the loop sequence, diminished binding of purified T4 DNA polymerase to its RNA in vitro (as measured by a gel retardation assay) and derepressed synthesis of the enzyme in vivo (as measured in T4 infections and by recombinant-plasmid-mediated expression). In vitro effects, however, were not always congruent with in vivo effects. For example, stem pairing with a sequence other than wild-type resulted in normal protein binding in vitro but derepression of protein synthesis in vivo. Similarly, a C----A change in the loop had a small effect in vitro and a strong effect in vivo. In contrast, an A----U change near the base of the hairpin that was predicted to increase the length of the base-paired stem had small effects both in vitro and in vivo. The results suggest that interaction of T4 DNA polymerase with its structured RNA operator depends on the spatial arrangement of specific nucleotide residues and is subject to modulation in vivo.

scholarly journals Mutational Analysis of Residues in PriA and PriC Affecting Their Ability To Interact with SSB in Escherichia coli K-12

2020 ◽  
Vol 202 (23) ◽  
Author(s):  
Anastasiia N. Klimova ◽  
Steven J. Sandler
Keyword(s):  
Escherichia Coli ◽  
Mutational Analysis ◽  
Wild Type ◽  
Content Type ◽  
Growth Defects ◽  
C Terminus ◽  
K 12 ◽  
And Function

ABSTRACT Escherichia coli PriA and PriC recognize abandoned replication forks and direct reloading of the DnaB replicative helicase onto the lagging-strand template coated with single-stranded DNA-binding protein (SSB). Both PriA and PriC have been shown by biochemical and structural studies to physically interact with the C terminus of SSB. In vitro, these interactions trigger remodeling of the SSB on ssDNA. priA341(R697A) and priC351(R155A) negated the SSB remodeling reaction in vitro. Plasmid-carried priC351(R155A) did not complement priC303::kan, and priA341(R697A) has not yet been tested for complementation. Here, we further studied the SSB-binding pockets of PriA and PriC by placing priA341(R697A), priA344(R697E), priA345(Q701E), and priC351(R155A) on the chromosome and characterizing the mutant strains. All three priA mutants behaved like the wild type. In a ΔpriB strain, the mutations caused modest increases in SOS expression, cell size, and defects in nucleoid partitioning (Par−). Overproduction of SSB partially suppressed these phenotypes for priA341(R697A) and priA344(R697E). The priC351(R155A) mutant behaved as expected: there was no phenotype in a single mutant, and there were severe growth defects when this mutation was combined with ΔpriB. Analysis of the priBC mutant revealed two populations of cells: those with wild-type phenotypes and those that were extremely filamentous and Par− and had high SOS expression. We conclude that in vivo, priC351(R155A) identified an essential residue and function for PriC, that PriA R697 and Q701 are important only in the absence of PriB, and that this region of the protein may have a complicated relationship with SSB. IMPORTANCE Escherichia coli PriA and PriC recruit the replication machinery to a collapsed replication fork after it is repaired and needs to be restarted. In vitro studies suggest that the C terminus of SSB interacts with certain residues in PriA and PriC to recruit those proteins to the repaired fork, where they help remodel it for restart. Here, we placed those mutations on the chromosome and tested the effect of mutating these residues in vivo. The priC mutation completely abolished function. The priA mutations had no effect by themselves. They did, however, display modest phenotypes in a priB-null strain. These phenotypes were partially suppressed by SSB overproduction. These studies give us further insight into the reactions needed for replication restart.

scholarly journals Analysis of YfgL and YaeT Interactions through Bioinformatics, Mutagenesis, and Biochemistry

2007 ◽  
Vol 190 (5) ◽  
pp. 1507-1517 ◽  
Author(s):  
Phu Vuong ◽  
Drew Bennion ◽  
Jeremy Mantei ◽  
Danielle Frost ◽  
Rajeev Misra
Keyword(s):  
Escherichia Coli ◽  
Steady State ◽  
Membrane Protein ◽  
Direct Contact ◽  
Mutational Analysis ◽  
Cross Linking ◽  
Conserved Residues ◽  
Protein Biogenesis ◽  
Steady State Levels

ABSTRACT In Escherichia coli, YaeT, together with four lipoproteins, YfgL, YfiO, NlpB, and SmpA, forms a complex that is essential for β-barrel outer membrane protein biogenesis. Data suggest that YfgL and YfiO make direct but independent physical contacts with YaeT. Whereas the YaeT-YfiO interaction needs NlpB and SmpA for complex stabilization, the YaeT-YfgL interaction does not. Using bioinformatics, genetics, and biochemical approaches, we have identified three residues, L173, L175, and R176, in the mature YfgL protein that are critical for both function and interactions with YaeT. A single substitution at any of these sites produces no phenotypic defect, but two or three simultaneous alterations produce mild or yfgL-null phenotypes, respectively. Interestingly, biochemical data show that all YfgL variants, including those with single substitutions, have weakened in vivo YaeT-YfgL interaction. These defects are not due to mislocalization or low steady-state levels of YfgL. Cysteine-directed cross-linking data show that the region encompassing L173, L175, and R176 makes direct contact with YaeT. Using the same genetic and biochemical strategies, it was found that altering residues D227 and D229 in another region of YfgL from E221 to D229 resulted in defective YaeT bindings. In contrast, mutational analysis of conserved residues V319 to H328 of YfgL shows that they are important for YfgL biogenesis but not YfgL-YaeT interactions. The five YfgL mutants defective in YaeT associations and the yfgL background were used to show that SurA binds to YaeT (or another complex member) without going through YfgL.

scholarly journals Analysis of the Role oftrans-Translation in the Requirement of tmRNA for λimmP22 Growth in Escherichia coli

1999 ◽  
Vol 181 (7) ◽  
pp. 2148-2157 ◽  
Author(s):  
Jeffrey Withey ◽  
David Friedman
Keyword(s):  
Escherichia Coli ◽  
Dna Binding ◽  
Binding Proteins ◽  
Mutational Analysis ◽  
Dna Binding Proteins ◽  
Peptide Sequence ◽  
Primary Role ◽  
Translation Model ◽  
Small Stable Rna

ABSTRACT The small, stable RNA molecule encoded by ssrA, known as tmRNA or 10Sa RNA, is required for the growth of certain hybrid λimm P22 phages in Escherichia coli. tmRNA has been shown to tag partially synthesized proteins for degradation in vivo by attaching a short peptide sequence, encoded by tmRNA, to the carboxyl termini of these proteins. This tag sequence contains, at its C terminus, an amino acid sequence that is recognized by cellular proteases and leads to degradation of tagged proteins. A model describing this function of tmRNA, thetrans-translation model (K. C. Keiler, P. R. Waller, and R. T. Sauer, Science 271:990–993, 1996), proposes that tmRNA acts first as a tRNA and then as a mRNA, resulting in release of the original mRNA template from the ribosome and translocation of the nascent peptide to tmRNA. Previous work from this laboratory suggested that tmRNA may also interact specifically with DNA-binding proteins, modulating their activity. However, more recent results indicate that interactions between tmRNA and DNA-binding proteins are likely nonspecific. In light of this new information, we examine the effects on λimm P22 growth of mutations eliminating activities postulated to be important for two different steps in the trans-translation model, alanine charging of tmRNA and degradation of tagged proteins. This mutational analysis suggests that, while charging of tmRNA with alanine is essential for λimm P22 growth in E. coli, degradation of proteins tagged by tmRNA is required only to achieve optimal levels of phage growth. Based on these results, we propose that trans-translation may have two roles, the primary role being the release of stalled ribosomes from their mRNA template and the secondary role being the tagging of truncated proteins for degradation.

scholarly journals A Mutation within the β Subunit of Escherichia coli RNA Polymerase Impairs Transcription from Bacteriophage T4 Middle Promoters

2010 ◽  
Vol 192 (21) ◽  
pp. 5580-5587 ◽  
Author(s):  
Tamara D. James ◽  
Michael Cashel ◽  
Deborah M. Hinton
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Bacteriophage T4 ◽  
Β Subunit ◽  
Subunit Gene ◽  
Wild Type ◽  
Promoter Activation ◽  
E Coli ◽  
Growth Phenotype

ABSTRACT During infection of Escherichia coli, bacteriophage T4 usurps the host transcriptional machinery, redirecting it to the expression of early, middle, and late phage genes. Middle genes, whose expression begins about 1 min postinfection, are transcribed both from the extension of early RNA into middle genes and by the activation of T4 middle promoters. Middle-promoter activation requires the T4 transcriptional activator MotA and coactivator AsiA, which are known to interact with σ70, the specificity subunit of RNA polymerase. T4 motA amber [motA(Am)] or asiA(Am) phage grows poorly in wild-type E. coli. However, previous work has found that T4 motA(Am)does not grow in the E. coli mutant strain TabG. We show here that the RNA polymerase in TabG contains two mutations within its β-subunit gene: rpoB(E835K) and rpoB(G1249D). We find that the G1249D mutation is responsible for restricting the growth of either T4 motA(Am)or asiA(Am) and for impairing transcription from MotA/AsiA-activated middle promoters in vivo. With one exception, transcription from tested T4 early promoters is either unaffected or, in some cases, even increases, and there is no significant growth phenotype for the rpoB(E835K G1249D) strain in the absence of T4 infection. In reported structures of thermophilic RNA polymerase, the G1249 residue is located immediately adjacent to a hydrophobic pocket, called the switch 3 loop. This loop is thought to aid in the separation of the RNA from the DNA-RNA hybrid as RNA enters the RNA exit channel. Our results suggest that the presence of MotA and AsiA may impair the function of this loop or that this portion of the β subunit may influence interactions among MotA, AsiA, and RNA polymerase.

scholarly journals Differential Mechanisms of Binding of Anti-Sigma Factors Escherichia coli Rsd and Bacteriophage T4 AsiA to E. coli RNA Polymerase Lead to Diverse Physiological Consequences

2008 ◽  
Vol 190 (10) ◽  
pp. 3434-3443 ◽  
Author(s):  
Umender K. Sharma ◽  
Dipankar Chatterji
Keyword(s):  
Escherichia Coli ◽  
Cell Growth ◽  
Rna Polymerase ◽  
Bacteriophage T4 ◽  
Sigma Factors ◽  
E Coli ◽  
Yeast Two Hybrid System ◽  
Vitro Binding

ABSTRACT Anti-sigma factors Escherichia coli Rsd and bacteriophage T4 AsiA bind to the essential housekeeping sigma factor, σ70, of E. coli. Though both factors are known to interact with the C-terminal region of σ70, the physiological consequences of these interactions are very different. This study was undertaken for the purpose of deciphering the mechanisms by which E. coli Rsd and bacteriophage T4 AsiA inhibit or modulate the activity of E. coli RNA polymerase, which leads to the inhibition of E. coli cell growth to different amounts. It was found that AsiA is the more potent inhibitor of in vivo transcription and thus causes higher inhibition of E. coli cell growth. Measurements of affinity constants by surface plasmon resonance experiments showed that Rsd and AsiA bind to σ70 with similar affinity. Data obtained from in vivo and in vitro binding experiments clearly demonstrated that the major difference between AsiA and Rsd is the ability of AsiA to form a stable ternary complex with RNA polymerase. The binding patterns of AsiA and Rsd with σ70 studied by using the yeast two-hybrid system revealed that region 4 of σ70 is involved in binding to both of these anti-sigma factors; however, Rsd interacts with other regions of σ70 as well. Taken together, these results suggest that the higher inhibition of E. coli growth by AsiA expression is probably due to the ability of the AsiA protein to trap the holoenzyme RNA polymerase rather than its higher binding affinity to σ70.

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