scholarly journals The Ribosomal Protein L2 Interacts with the RNA Polymerase α Subunit and Acts as a Transcription Modulator in Escherichia coli

2010 ◽  
Vol 192 (7) ◽  
pp. 1882-1889 ◽  
Author(s):  
Valentina Rippa ◽  
Claudia Cirulli ◽  
Benedetta Di Palo ◽  
Nunzianna Doti ◽  
Angela Amoresano ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Ribosomal Protein ◽  
Transcription Initiation ◽  
Direct Interaction ◽  
Regulation Of Transcription ◽  
Interacting Proteins ◽  
Α Subunit ◽  
Gene Reporter Assay ◽  
Ribosomal Protein L2

ABSTRACT Identification of interacting proteins in stable complexes is essential to understand the mechanisms that regulate cellular processes at the molecular level. Transcription initiation in prokaryotes requires coordinated protein-protein and protein-DNA interactions that often involve one or more transcription factors in addition to RNA polymerase (RNAP) subunits. The RNAP α subunit (RNAPα) is a key regulatory element in gene transcription and functions through direct interaction with other proteins to control all stages of this process. A clear description of the RNAPα protein partners should greatly increase our understanding of transcription modulation. A functional proteomics approach was employed to investigate protein components that specifically interact with RNAPα. A tagged form of Escherichia coli RNAPα was used as bait to determine the molecular partners of this subunit in a whole-cell extract. Among other interacting proteins, 50S ribosomal protein L2 (RPL2) was clearly identified by mass spectrometry. The direct interaction between RNAPα and RPL2 was confirmed both in vivo and in vitro by performing coimmunoprecipitation and bacterial two-hybrid experiments. The functional role of this interaction was also investigated in the presence of a ribosomal promoter by using a β-galactosidase gene reporter assay. The results clearly demonstrated that RPL2 was able to increase β-galactosidase expression only in the presence of a specific ribosomal promoter, whereas it was inactive when it was assayed with an unrelated promoter. Interestingly, other ribosomal proteins (L1, L3, L20, and L27) did not have any effect on rRNA expression. The findings reported here strongly suggest that in addition to its role in ribosome assembly the highly conserved RPL2 protein plays a specific and direct role in regulation of transcription.

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.

Activation by TyrR in Escherichia coli K-12 by Interaction between TyrR and the α-subunit of RNA polymerase

2021 ◽  
Author(s):  
Helen Camakaris ◽  
Ji Yang ◽  
Tadashi Fujii ◽  
James Pittard
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Regulatory Protein ◽  
Aromatic Amino Acids ◽  
Regulation Of Transcription ◽  
Plant Interactions ◽  
Α Subunit ◽  
Partial Suppression ◽  
K 12

A novel selection was developed for RpoA α-CTD mutants altered in activation by the TyrR regulatory protein of E. coli K-12. This allowed the identification of an aspartate to asparagine substitution in residue 250 (DN250) as an Act - mutation. Amino acid residues known to be close to D250 were altered by in vitro mutagenesis, and substitutions DR250, RE310 and RD310 were all shown to be defective in activation. None of these mutations caused defects in UP regulation. The rpoA mutation DN250 was transferred onto the chromosome to facilitate the isolation of suppressor mutations. TyrR Mutations EK139 and RG119 caused partial suppression of rpoA DN250, and TyrR RC119, RL119, RP119, RA77 and SG100 caused partial suppression of rpoA RE310. Additional activation-defective rpoA mutants (DT250, RS310, EG288) were also isolated, using the chromosomal rpoA DN250 strain. Several new Act - tyrR mutants were isolated in an rpoA + strain, adding positions R77, D97, K101, D118, R119, R121 and E141 to known residues, S95 and D103, and defining the ‘activation patch’ on the NTD of TyrR. These results support a model for activation of TyrR-regulated genes where the ‘activation patch’ on the TyrR NTD interacts with the ‘TyrR-specific patch’ on the αCTD of RNA polymerase. Given known structures, both these sites appear to be surface exposed, and suggest a model for activation by TyrR. They also help resolve confusing results in the literature that implicated residues within the 261 and 265 determinants, as Activator contact sites. IMPORTANCE Regulation of transcription by RNA polymerases is fundamental for adaptation to a changing environment and for cellular differentiation, across all kingdoms of life. The gene TyrR in Escherichia coli is a particularly useful model because it is involved in both activation and repression of a large number of operons by a range of mechanisms, and it interacts with all three aromatic amino acids and probably other effectors. Furthermore TyrR has homologues in many other genera, regulating many different genes, utilizing different effector molecules, and in some cases affecting virulence, and important plant interactions.

scholarly journals Activation by NarL at the Escherichia coli ogt promoter

2020 ◽  
Vol 477 (15) ◽  
pp. 2807-2820
Author(s):  
Patcharawarin Ruanto ◽  
David L. Chismon ◽  
Joanne Hothersall ◽  
Rita E. Godfrey ◽  
David J. Lee ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Response Regulator ◽  
Direct Interaction ◽  
Α Subunit ◽  
E Coli ◽  
Terminal Domain ◽  
Dna Alkyltransferase ◽  
Promoter Dna ◽  
Transcript Initiation

The Escherichia coli NarX/NarL two-component response-regulator system regulates gene expression in response to nitrate ions and the NarL protein is a global transcription factor, which activates transcript initiation at many target promoters. One such target, the E. coli ogt promoter, which controls the expression of an O6-alkylguanine-DNA-alkyltransferase, is dependent on NarL binding to two DNA targets centred at positions −44.5 and −77.5 upstream from the transcript start. Here, we describe ogt promoter derivatives that can be activated solely by NarL binding either at position −44.5 or position −77.5. We show that NarL can also activate the ogt promoter when located at position −67.5. We present data to argue that NarL-dependent activation of transcript initiation at the ogt promoter results from a direct interaction between NarL and a determinant in the C-terminal domain of the RNA polymerase α subunit. Footprinting experiments show that, at the −44.5 promoter, NarL and the C-terminal domain of the RNA polymerase α subunit bind to opposite faces of promoter DNA, suggesting an unusual mechanism of transcription activation. Our work suggests new organisations for activator-dependent transcription at promoters and future applications for biotechnology.

scholarly journals Spacing requirements for interactions between the C-terminal domain of the α subunit of Escherichia coli RNA polymerase and the cAMP receptor protein

1998 ◽  
Vol 330 (1) ◽  
pp. 413-420 ◽  
Author(s):  
S. Georgina LLOYD ◽  
J. W. Stephen BUSBY ◽  
J. Nigel SAVERY
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Binding Site ◽  
Transcription Initiation ◽  
Receptor Protein ◽  
Camp Receptor Protein ◽  
Α Subunit ◽  
Synthetic Promoters ◽  
Terminal Domain ◽  
Camp Receptor

During transcription initiation at bacterial promoters, the C-terminal domain of the RNA polymerase α subunit (αCTD) can interact with DNA-sequence elements (known as UP elements) and with activator proteins. We have constructed a series of semi-synthetic promoters carrying both an UP element and a consensus DNA-binding site for the Escherichia coli cAMP receptor protein (CRP; a factor that activates transcription by making direct contacts with αCTD). At these promoters, the UP element was located at a variety of distances upstream of the CRP-binding site, which was fixed at position -41.5 bp upstream of the transcript start. At some positions, the UP element caused enhanced promoter activity whereas, at other positions, it had very little effect. In no case was the CRP-dependence of the promoter relieved. DNase I and hydroxyl-radical footprinting were used to study ternary RNA polymerase-CRP-promoter complexes formed at two of the most active of these promoters, and co-operativity between the binding of CRP and purified α subunits was studied. The footprints show that αCTD binds to the UP element as it is displaced upstream but that this displacement does not prevent αCTD from being contacted by CRP. Models to account for this are discussed.

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.

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