Tamas Gaal

UPs and downs in bacterial transcription initiation: the role of the alpha subunit of RNA polymerase in promoter recognition

In recent years, it has become clear that promoter recognition by bacterial RNA polymerase involves interactions not only between core promoter elements and the σ subunit, but also between a DNA element upstream of the core promoter and the α subunit. DNA binding by α can increase transcription dramatically. Here we review the current state of our understanding of the α interaction with DNA during basal transcription initiation (i.e. in the absence of proteins other than RNA polymerase) and activated transcription initiation (i.e. when stimulated by transcription factors).

Promoter recognition and discrimination by EsigmaS RNA polymerase.

Although more than 30 Escherichia coli promoters utilize the RNA polymerase holoenzyme containing σS (EσS), and it is known that there is some overlap between the promoters recognized by EσS and by the major E. coli holoenzyme (Eσ70), the sequence elements responsible for promoter recognition by EσS are not well understood. To define the DNA sequences recognized best by EσSin vitro, we started with random DNA and enriched for EσS promoter sequences by multiple cycles of binding and selection. Surprisingly, the sequences selected by EσS contained the known consensus elements (−10 and −35 hexamers) for recognition by Eσ70. Using genetic and biochemical approaches, we show that EσS and Eσ70 do not achieve specificity through ‘best fit’ to different consensus promoter hexamers, the way that other forms of holoenzyme limit transcription to discrete sets of promoters. Rather, we suggest that EσS‐specific promoters have sequences that differ significantly from the consensus in at least one of the recognition hexamers, and that promoter discrimination against Eσ70 is achieved, at least in part, by the two enzymes tolerating different deviations from consensus. DNA recognition by EσS versus Eσ70 thus presents an alternative solution to the problem of promoter selectivity.

Transcription activation at Class II CRP-dependent promoters: identification of determinants in the C-terminal domain of the RNA polymerase alpha subunit.

Many transcription factors, including the Escherichia coli cyclic AMP receptor protein (CRP), act by making direct contacts with RNA polymerase. At Class II CRP‐dependent promoters, CRP activates transcription by making two such contacts: (i) an interaction with the RNA polymerase α subunit C‐terminal domain (αCTD) that facilitates initial binding of RNA polymerase to promoter DNA; and (ii) an interaction with the RNA polymerase α subunit N‐terminal domain that facilitates subsequent promoter opening. We have used random mutagenesis and alanine scanning to identify determinants within αCTD for transcription activation at a Class II CRP‐dependent promoter. Our results indicate that Class II CRP‐dependent transcription requires the side chains of residues 265, 271, 285–288 and 317. Residues 285–288 and 317 comprise a discrete 20×10 Å surface on αCTD, and substitutions within this determinant reduce or eliminate cooperative interactions between α subunits and CRP, but do not affect DNA binding by α subunits. We propose that, in the ternary complex of RNA polymerase, CRP and a Class II CRP‐dependent promoter, this determinant in αCTD interacts directly with CRP, and is distinct from and on the opposite face to the proposed determinant for αCTD–CRP interaction in Class I CRP‐dependent transcription.

Molecular anatomy of a transcription activation patch: FIS–RNA polymerase interactions at the Escherichia coli rrnB P1 promoter

FIS, a site‐specific DNA binding and bending protein, is a global regulator of gene expression in Escherichia coli. The ribosomal RNA promoter rrnB P1 is activated 3‐ to 7‐fold in vivo by a FIS dimer that binds a DNA site immediately upstream of the DNA binding site for the C‐terminal domain (CTD) of the α subunit of RNA polymerase (RNAP). In this report, we identify several FIS side chains important specifically for activation of transcription at rrnB P1. These side chains map to positions 68, 71 and 74, in and flanking a surface‐exposed loop adjacent to the helix–turn–helix DNA binding motif of the protein. We also present evidence suggesting that FIS activates transcription at rrnB P1 by interacting with the RNAP αCTD. Our results suggest a model for FIS‐mediated activation of transcription at rrnB P1 that involves interactions between FIS and the RNAP αCTD near the DNA surface. Although FIS and the transcription activator protein CAP have little structural similarity, they both bend DNA, use a similarly disposed activation loop and target the same region of the RNAP αCTD, suggesting that this is a common architecture at bacterial promoters.

NTP-sensing by rRNA promoters in Escherichia coli is direct

We showed previously that rrn P1 promoters require unusually high concentrations of the initiating nucleoside triphosphates (ATP or GTP, depending on the promoter) for maximal transcription in vitro. We proposed that this requirement for high initiating NTP concentrations contributes to control of the rrn P1 promoters from the seven Escherichia coli rRNA operons. However, the previous studies did not prove that variation in NTP concentration affects rrn P1 promoter activity directly in vivo. Here, we create conditions in vivo in which ATP and GTP concentrations are altered in opposite directions relative to one another, and we show that transcription from rrn P1 promoters that initiate with either ATP or GTP follows the concentration of the initiating NTP for that promoter. These results demonstrate that the effect of initiating NTP concentration on rrn P1 promoter activity in vivo is direct. As predicted by a model in which homeostatic control of rRNA transcription results, at least in part, from sensing of NTP concentrations by rrn P1 promoters, we show that inhibition of protein synthesis results in an increase in ATP concentration and a corresponding increase in transcription from rrnB P1. We conclude that translation is a major consumer of purine NTPs, and that NTP-sensing by rrn P1 promoters serves as a direct regulatory link between translation and ribosome synthesis.

Still looking for the magic spot: the crystallographically-defined binding site for ppGpp on RNA polymerase is unlikely to be responsible for rRNA transcription regulation

Identification of the RNA polymerase (RNAP) binding site for ppGpp, a central regulator of bacterial transcription, is crucial for understanding its mechanism of action. A recent high-resolution X-ray structure defined a ppGpp binding site on Thermus thermophilus RNAP. We report here effects of ppGpp on 10 mutant Escherichia coli RNAPs with substitutions for the analogous residues within 3-4 A of the ppGpp binding site in the T. thermophilus cocrystal. None of the substitutions in E. coli RNAP significantly weakened its responses to ppGpp. This result differs from the originally reported finding of a substitution in E. coli RNAP eliminating ppGpp function. The E. coli RNAPs used in that study likely lacked stoichiometric amounts of omega, an RNAP subunit required for responses of RNAP to ppGpp, in part explaining the discrepancy. Furthermore, we found that ppGpp did not inhibit transcription initiation by T. thermophilus RNAP in vitro or shorten the lifetimes of promoter complexes containing T. thermophilus RNAP, in contrast to the conclusion in the original report. Our results suggest that the ppGpp binding pocket identified in the cocrystal is not the one responsible for regulation of E. coli ribosomal RNA transcription initiation and highlight the importance of inclusion of omega in bacterial RNAP preparations.

Crl Facilitates RNA Polymerase Holoenzyme Formation

The Escherichia coli Crl protein has been described as a transcriptional coactivator for the stationary-phase sigma factor sigma(S). In a transcription system with highly purified components, we demonstrate that Crl affects transcription not only by the Esigma(S) RNA polymerase holoenzyme but also by Esigma(70) and Esigma(32). Crl increased transcription dramatically but only when the sigma concentration was low and when Crl was added to sigma prior to assembly with the core enzyme. Our results suggest that Crl facilitates holoenzyme formation, the first positive regulator identified with this mechanism of action.

Sequences upstream of the −35 hexamer of rrnB P1 affect promoter strength and upstream activation

Transcription from Escherichia coli ribosomal RNA promoters is increased about 20-fold in vivo by a DNA sequence (the Upstream Activation Region, UAR) located upstream of the -35 conserved hexamer. The UAR stimulates transcription through two mechanisms: one which involves binding of the Fis protein to the UAR, and another mechanisms which functions in the absence of additional protein factors. We have previously constructed a collection of mutations in the region upstream of the -35 hexamer of rrnB P1. Most of these mutations have either no effect on promoter activity or decrease activity 2-5-fold in vivo (Gaal, T., Barkei, J., Dickson, R.R., De Boer, H.A., De Haseth, P.L., Alavi, H. and Gourse, R.L.(1989) J. Bacteriol. 171, 4852-4861). Two mutations leave both the -35 consensus hexamer and the Fis binding consensus sequence intact, yet have larger (14-50-fold) effects on transcription. One substitution just upstream of the -35 hexamer (a C to T change at position -37) primarily affects intrinsic promoter strength, leaving the UAR functional. On the other hand, a three base pair deletion (bases -38 through -40) severely reduces UAR-mediated activity. A substitution covering the three base pair deletion was constructed and found to be activated normally. UAR function appears dependent on its position relative to the RNA polymerase binding site, suggesting that a particular spatial geometry may be necessary for Fis-dependent and/or factor-independent activation to occur.

Transcription Activation by CooA, the CO-sensing Factor fromRhodospirillum rubrum THE INTERACTION BETWEEN CooA AND THE C-TERMINAL DOMAIN OF THE α SUBUNIT OF RNA POLYMERASE

CooA, a member of the cAMP receptor protein (CRP) family, is a CO-sensing transcription activator fromRhodospirillum rubrum that binds specific DNA sequences in response to CO. The location of the CooA-binding sites relative to the start sites of transcription suggested that the CooA-dependent promoters are analogous to class II CRP-dependent promoters. In this study, we developed anin vivo CooA reporter system in Escherichia coli and an in vitro transcription assay using RNA polymerases (RNAP) from E. coli and from Rhodobacter sphaeroides to study the transcription properties of CooA and the protein-protein interaction between CooA and RNAP. The ability of CooA to activate CO-dependent transcription in vivoin heterologous backgrounds suggested that CooA is sufficient to direct RNAP to initiate transcription and that no other factors are required. This hypothesis was confirmed in vitro with purified CooA and purified RNAP. Use of a mutant form of E. coli RNAP with α subunits lacking their C-terminal domain (α-CTD) dramatically decreased CooA-dependent transcription of the CooA-regulated R. rubrum promoter P cooF in vitro, which indicates that α-CTD plays an important role in this activation. DNase I footprinting analysis showed that CooA facilitates binding of wild-type RNAP, but not α-CTD-truncated RNAP, to P cooF . This facilitated binding provides evidence for a direct contact between CooA and α-CTD of RNAP during activation of transcription. Mapping the CooA-contact site in α-CTD suggests that CooA is similar but not identical to CRP in terms of its contact sites to the α-CTD at class II promoters.

Escherichia coli DksA Binds to Free RNA Polymerase with Higher Affinity than to RNA Polymerase in an Open Complex

The transcription factor DksA binds in the secondary channel of RNA polymerase (RNAP) and alters transcriptional output without interacting with DNA. Here we present a quantitative assay for measuring DksA binding affinity and illustrate its utility by determining the relative affinities of DksA for three different forms of RNAP. Whereas the apparent affinities of DksA for RNAP core and holoenzyme are the same, the apparent affinity of DksA for RNAP decreases almost 10-fold in an open complex. These results suggest that the conformation of RNAP present in an open complex is not optimal for DksA binding and that DNA directly or indirectly alters the interface between the two proteins.