scholarly journals A mismatch bubble in double-stranded DNA suffices to direct precise transcription initiation by Escherichia coli RNA polymerase.

1994 ◽  
Vol 269 (18) ◽  
pp. 13179-13184
Author(s):  
S.E. Aiyar ◽  
J.D. Helmann ◽  
P.L. deHaseth
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Double Stranded Dna

scholarly journals Interactions between the Escherichia coli cAMP receptor protein and the C-terminal domain of the α subunit of RNA polymerase at Class I promoters

1999 ◽  
Vol 337 (3) ◽  
pp. 415-423 ◽  
Author(s):  
Emma C. LAW ◽  
Nigel J. SAVERY ◽  
Stephen J. W. BUSBY
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Class I ◽  
Receptor Protein ◽  
Camp Receptor Protein ◽  
Α Subunit ◽  
Terminal Domain ◽  
Up Elements ◽  
Camp Receptor

The Escherichia coli cAMP receptor protein (CRP) is a factor that activates transcription at over 100 target promoters. At Class I CRP-dependent promoters, CRP binds immediately upstream of RNA polymerase and activates transcription by making direct contacts with the C-terminal domain of the RNA polymerase α subunit (αCTD). Since αCTD is also known to interact with DNA sequence elements (known as UP elements), we have constructed a series of semi-synthetic Class I CRP-dependent promoters, carrying both a consensus DNA-binding site for CRP and a UP element at different positions. We previously showed that, at these promoters, the CRP–αCTD interaction and the CRP–UP element interaction contribute independently and additively to transcription initiation. In this study, we show that the two halves of the UP element can function independently, and that, in the presence of the UP element, the best location for the DNA site for CRP is position -69.5. This suggests that, at Class I CRP-dependent promoters where the DNA site for CRP is located at position -61.5, the two αCTDs of RNA polymerase are not optimally positioned. Two experiments to test this hypothesis are presented.

scholarly journals Effects of distortions by A-tracts of promoter B-DNA spacer region on the kinetics of open complex formation by Escherichia coli RNA polymerase.

2003 ◽  
Vol 50 (4) ◽  
pp. 909-920 ◽  
Author(s):  
Iwona K Kolasa ◽  
Tomasz Łoziński ◽  
Kazimierz L Wierzchowski
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Specific Interaction ◽  
Structural Data ◽  
Structural Morphology ◽  
Promoter Dna ◽  
Sigma Subunit ◽  
Kinetics Of

A-tracts in DNA due to their structural morphology distinctly different from the canonical B-DNA form play an important role in specific recognition of bacterial upstream promoter elements by the carboxyl terminal domain of RNA polymerase alpha subunit and, in turn, in the process of transcription initiation. They are only rarely found in the spacer promoter regions separating the -35 and -10 recognition hexamers. At present, the nature of the protein-DNA contacts formed between RNA polymerase and promoter DNA in transcription initiation can only be inferred from low resolution structural data and mutational and crosslinking experiments. To probe these contacts further, we constructed derivatives of a model Pa promoter bearing in the spacer region one or two An (n = 5 or 6) tracts, in phase with the DNA helical repeat, and studied the effects of thereby induced perturbation of promoter DNA structure on the kinetics of open complex (RPo) formation in vitro by Escherichia coli RNA polymerase. We found that the overall second-order rate constant ka of RPo formation, relative to that at the control promoter, was strongly reduced by one to two orders of magnitude only when the A-tracts were located in the nontemplate strand. A particularly strong 30-fold down effect on ka was exerted by nontemplate A-tracts in the -10 extended promoter region, where an involvement of nontemplate TG (-14, -15) sequence in a specific interaction with region 3 of sigma-subunit is postulated. A-tracts in the latter location caused also 3-fold slower isomerization of the first closed transcription complex into the intermediate one that precedes formation of RPo, and led to two-fold faster dissociation of the latter. All these findings are discussed in relation to recent structural and kinetic models of RPo formation.

scholarly journals Affinity Isolation and I-DIRT Mass Spectrometric Analysis of the Escherichia coli O157:H7 Sakai RNA Polymerase Complex

2007 ◽  
Vol 190 (4) ◽  
pp. 1284-1289 ◽  
Author(s):  
David J. Lee ◽  
Stephen J. W. Busby ◽  
Lars F. Westblade ◽  
Brian T. Chait
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Laboratory Strain ◽  
Effector Proteins ◽  
Spectrometric Analysis ◽  
Isolation Technique ◽  
E Coli ◽  
Affinity Isolation ◽  
K 12

ABSTRACT Bacteria contain a single multisubunit RNA polymerase that is responsible for the synthesis of all RNA. Previous studies of the Escherichia coli K-12 laboratory strain identified a group of effector proteins that interact directly with RNA polymerase to modulate the efficiency of transcription initiation, elongation, or termination. Here we used a rapid affinity isolation technique to isolate RNA polymerase from the pathogenic Escherichia coli strain O157:H7 Sakai. We analyzed the RNA polymerase enzyme complex using mass spectrometry and identified associated proteins. Although E. coli O157:H7 Sakai contains more than 1,600 genes not present in the K-12 strain, many of which are predicted to be involved in transcription regulation, all of the identified proteins in this study were encoded on the “core” E. coli genome.

scholarly journals Translational Regulation of the Escherichia coli Stress Factor RpoS: a Role for SsrA and Lon

2007 ◽  
Vol 189 (13) ◽  
pp. 4872-4879 ◽  
Author(s):  
Caroline Ranquet ◽  
Susan Gottesman
Keyword(s):  
Escherichia Coli ◽  
Low Temperature ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Translational Regulation ◽  
Regulatory Rna ◽  
Gene Encoding ◽  
Ribosome Stalling ◽  
High Level ◽  
Sigma Subunit

ABSTRACT Escherichia coli cell viability during starvation is strongly dependent on the expression of the rpoS gene, encoding the RpoS sigma subunit of RNA polymerase. RpoS abundance has been reported to be regulated at many levels, including transcription initiation, translation, and protein stability. The regulatory RNA SsrA (or tmRNA) has both tRNA and mRNA activities, relieving ribosome stalling and cotranslationally tagging proteins. We report here that SsrA is needed for the correct high-level translation of RpoS. The ATP-dependent protease Lon was also found to negatively affect RpoS translation, but only at low temperature. We suggest that SsrA may indirectly improve RpoS translation by limiting ribosome stalling and depletion of some component of the translation machinery.

scholarly journals A Novel Initiation Pathway in Escherichia Coli Transcription

2016 ◽  
Author(s):  
Eitan Lerner ◽  
SangYoon Chung ◽  
Benjamin L. Allen ◽  
Shuang Wang ◽  
Jookyung J. Lee ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Magnetic Tweezer ◽  
Delayed Initiation ◽  
Rate Limiting ◽  
Kinetics Of

AbstractInitiation is a highly regulated, rate-limiting step in transcription. We employed a series of approaches to examine the kinetics of RNA polymerase (RNAP) transcription initiation in greater detail. Quenched kinetics assays, in combination with magnetic tweezer experiments and other methods, showed that, contrary to expectations, RNAP exit kinetics from later stages of initiation (e.g. from a 7-base transcript) was markedly slower than from earlier stages. Further examination implicated a previously unidentified intermediate in which RNAP adopted a long-lived backtracked state during initiation. In agreement, the RNAP-GreA endonuclease accelerated transcription kinetics from otherwise delayed initiation states and prevented RNAP backtracking. Our results indicate a previously uncharacterized RNAP initiation state that could be exploited for therapeutic purposes and may reflect a conserved intermediate among paused, initiating eukaryotic enzymes.Significance:Transcription initiation by RNAP is rate limiting owing to many factors, including a newly discovered slow initiation pathway characterized by RNA backtracking and pausing. This backtracked and paused state occurs when all NTPs are present in equal amounts, but becomes more prevalent with NTP shortage, which mimics cellular stress conditions. Pausing and backtracking in initiation may play an important role in transcriptional regulation, and similar backtracked states may contribute to pausing among eukaryotic RNA polymerase II enzymes.

Escherichia coli RNA polymerase binding sites and transcription initiation sites in the transposon Tn3

Gene ◽  
1983 ◽  
Vol 24 (1) ◽  
pp. 99-113 ◽  
Author(s):  
William L. Wishart ◽  
Machida Chiyoko ◽  
Ohtsubo Hisako ◽  
Ohtsubo Eiichi
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Binding Sites ◽  
Transcription Initiation ◽  
Polymerase Binding

scholarly journals The organization of open complexes between Escherichia coli RNA polymerase and DNA fragments carrying promoters either with or without consensus −35 region sequences

1990 ◽  
Vol 270 (1) ◽  
pp. 141-148 ◽  
Author(s):  
B Chan ◽  
A Spassky ◽  
S Busby
Keyword(s):  
Escherichia Coli ◽  
Complex Formation ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Point Mutations ◽  
Dna Fragments ◽  
Transcription Start ◽  
Footprint Analysis ◽  
Open Complex Formation ◽  
Promoter Dna

Transcription initiation at the Escherichia coli galP1 promoter does not depend on specific nucleotide sequences in the -35 region. Footprint analysis of transcriptionally competent complexes between E. coli RNA polymerase and DNA fragments carrying galP1 shows that RNA polymerase protects sequences as far upstream as -55, whereas sequences around the -35 region are exposed. In contrast, with galP1 derivatives carrying -35 region sequences resembling the consensus, RNA polymerase protects bases as far as -45, and the -35 region is fully protected. Taken together, our data suggest that the overall architecture of RNA polymerase-promoter complexes can vary according to whether or not consensus -35 region sequences are present; in the absence of these sequences, open complex formation requires distortion of the promoter DNA. However, the unwinding of promoter DNA around the transcription start is not affected by the nature of the -35 region sequence. With a galP1 derivative carrying point mutations in the spacer region that greatly reduce promoter activity, the protection of bases by RNA polymerase around the -10 sequence and transcription start site is reduced. In contrast, protection of the region upstream of -25 is unaffected by the spacer mutations, although sequences from -46 to -54 become hypersensitive to attack by potassium permanganate, indicating severe distortion or kinking of this zone. We suggest that, with this galP1 derivative, RNA polymerase is blocked in a complex that is an intermediate on the path to open complex formation.

scholarly journals Thymine at —5 Is Crucial for cpc Promoter Activity of Synechocystis sp. Strain PCC 6714

2003 ◽  
Vol 185 (21) ◽  
pp. 6477-6480 ◽  
Author(s):  
Masahiko Imashimizu ◽  
Shoko Fujiwara ◽  
Ryohei Tanigawa ◽  
Kan Tanaka ◽  
Takatsugu Hirokawa ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Promoter Activity ◽  
Initiation Site ◽  
Pcc 7942 ◽  
Rna Polymerase Promoter ◽  
Synechocystis Sp

ABSTRACT The levels of transcripts of the cpc operon were highly reduced in a PD-1 mutant of cyanobacterium Synechocystis sp. strain PCC 6714. This was due to a substitution of C for T that occurred at 5 bp upstream of the transcription initiation site of the cpc operon. Any substitution for T at the −5 position drastically reduced both in vivo and in vitro promoter activity in cyanobacterium Synechococcus sp. strain PCC 7942 but not the in vivo activity in Escherichia coli. This suggests that the requirement of −5T appears to be specific for a cyanobacterial RNA polymerase-promoter combination.

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