The σ70 subunit of RNA polymerase induces lacUV5 promoter-proximal pausing of transcription (original) (raw)
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The σ70 subunit of RNA polymerase mediates a promoter-proximal pause at the lac promoter
Nature Structural & Molecular Biology, 2004
The 70 subunit of RNA polymerase plays an essential role in transcription initiation. In addition, 70 has a critical regulatory role during transcription elongation at the bacteriophage late promoter, P R′ . At this promoter, 70 mediates a pause in early elongation through contact with a DNA sequence element in the initially transcribed region that resembles a promoter -10 element. Here we provide evidence that 70 also mediates a pause in early elongation at the lac promoter (plac). Like that at P R′ , the pause at plac is facilitated by a sequence element in the initially transcribed region that resembles a promoter -10 element. Using biophysical analysis, we demonstrate that the pause-inducing sequence element at plac stabilizes the interaction between 70 and the remainder of the transcription elongation complex. Bioinformatic analysis suggests that promoter-proximal 70 -dependent pauses may play a role in the regulation of many bacterial promoters.
Proceedings of the National Academy of Sciences, 2005
The -subunit of bacterial RNA polymerase (RNAP) is required for promoter-specific transcription initiation. This function depends on specific intersubunit interactions that occur when associates with the RNAP core enzyme to form RNAP holoenzyme. Among these interactions, that between conserved region 4 of and the flap domain of the RNAP -subunit (-flap) is critical for recognition of the major class of bacterial promoters. Here, we describe the isolation of amino acid substitutions in region 4 of Escherichia coli 70 that have specific effects on the 70 region 4͞-flap interaction, either weakening or strengthening it. Using these 70 mutants, we demonstrate that the region 4͞-flap interaction also can affect events occurring downstream of transcription initiation during early elongation. Specifically, our results provide support for a structure-based proposal that, when bound to the -flap, region 4 presents a barrier to the extension of the nascent RNA as it emerges from the RNA exit channel. Our findings support the view that the transition from initiation to elongation involves a staged disruption of -core interactions.
Interaction of RNA polymerase with lacUV5 promoter DNA during mRNA initiation and elongation
Journal of Molecular Biology, 1985
We have used enzymatic and chemical probes to follow the movement of Escherichia coli RNA polymerase along ZacUV5 promoter DNA during transcription initiation. The RNA polymerase does not escape from the promoter but remains tightly bound during the synthesis of the initial bases of the transcript. This initial phase of RNA synthesis involves the reiterative synthesis and release of RNA chains up to ten bases long via the R%A polymerase cycling reaction and the enzyme remains sensitive to rifampicin inhibition. When longer chains are made, promoter-specific binding is disrupted and the enzyme forms a rifampicin-resistant elongation complex with downstream DNA sequences. This elongation complex covers less than half as much DNA and lacks the DBase I-hypersensitive sites and the base-specific contacts that characterize promoter-bound RNA polymerase. These results lead us to suggest that ZucUV5 mRNA synthesis is primed by a promoter-bound enzyme complex that synthesizes the initial nine or ten bases in the mRNA chain. Subsequently, when a chain of ten bases, or slightly longer, is made. contacts with promoter DNA are irreversibly disrupted, sigma subunit is lost, and a "t'rur" elongation complex is formed.
Journal of Molecular Biology, 1998
The lac operon of Escherichia coli is positively regulated by the catabolite activator protein (CAP) bound upstream of the À45 region (CAP binding is centered at À61.5; the À45 region extends from À50 to À38). Certain mutations within the À45 region generate sequences that resemble UP elements in base composition and mimic the stimulation by the rrnBP1 UP element, yielding up to 15-fold stimulation in vivo. These À45 regioǹ`U P mutants'' are compromised in their CAP stimulation. CAP and UP elements do not act in a fully additive manner in vivo at the lac operon. Transcription assays with the wild-type lac promoter and an UP mutant of lac indicate that CAP and UP DNA also fail to act in a completely additive manner in vitro. RNA polymerase can stabilize CAP binding to promoter DNA with a À45 region UP element against a heparin challenge. This shows that CAP and the UP DNA do not compete for the a-CTD as a mechanism for their lack of additivity. CAP and UP elements both demonstrate decreased stimulation of transcription as RNA polymerase concentration is increased from 0.05 to 10 nM in in vitro transcription experiments. In addition CAP also stimulates transcription in a manner that does not decrease as RNA polymerase is varied over this concentration range. This invariable stimulation is by two-to threefold and occurs both in vivo and in vitro. It is not dependent upon the a-CTD of RNA polymerase and is maintained in the presence of the AR1 CAP mutant HL159. This two-to threefold invariable CAP stimulation appears to depend on the À45 region sequence as our À45 region mutants demonstrate different responses to HL159 CAP stimulation in vivo.
Molecular Cell, 2005
We report a single-molecule assay that defines, simultaneously, the translocational position of a protein complex relative to DNA and the subunit stoichiometry of the complex. We applied the assay to define translocational positions and s 70 contents of bacterial transcription elongation complexes in vitro. The results confirm ensemble results indicating that a large fraction, w70%-90%, of early elongation complexes retain s 70 and that a determinant for s 70 recognition in the initial transcribed region increases s 70 retention in early elongation complexes. The results establish that a significant fraction, w50%-60%, of mature elongation complexes retain s 70 and that a determinant for s 70 recognition in the initial transcribed region does not appreciably affect s 70 retention in mature elongation complexes. The results further establish that, in mature elongation complexes that retain s 70 , the half-life of s 70 retention is long relative to the timescale of elongation, suggesting that some complexes may retain s 70 throughout elongation.
Journal of Molecular Biology, 1998
The s subunit of RNA polymerase orchestrates basal transcription by ®rst binding to core RNA polymerase and then recognizing promoters. Using a series of 16 alanine-substitution mutations, we show that residues in a narrow region of Escherichia coli s 70 (590 to 603) are involved in transcription activation by a mutationally altered CRP derivative, FNR and AraC. Homology modeling of region 4 of s 70 to the closely related NarL or 434 Cro proteins, suggests that the ®ve basic residues implicated in activation are either in the C terminus of a long recognition helix that includes residues recognizing the À35 hexamer region of the promoter, or in the subsequent loop, and are ideally positioned to permit interaction with activators. The only substitution that has a signi®cant effect on activator-independent transcription is at R603, indicating that this residue of s 70 may play a distinct role in transcription initiation.
Journal of Molecular Biology, 2005
Transcription initiation in bacteria requires melting of w13 bp of promoter DNA. The mechanism of the melting process is not fully understood. Escherichia coli RNA polymerase bearing a deletion of the b subunit lobe I (amino acid residues 186-433) initiates melting of the K10 promoter element but cannot propagate the melting downstream, towards the transcription initiation start site (C1). However, in the presence of nucleotides, stable downstream melting is induced. Here, we studied lacUV5 promoter complexes formed by the mutant enzyme by crosslinking RNA polymerase subunits to single-stranded DNA in the transcription bubble. In the absence of NTPs, a contact between the s 70 subunit and the non-template strand of the K10 promoter element was detected. This contact disappeared in the presence of NTPs. Instead, a new s 70 -DNA contact as well as stable b 0 and b subunit contacts with the nontemplate DNA downstream of the K10 promoter element were established. In terms of the two-step (upstream initiation/downstream propagation) model of promoter melting, our data suggest that b lobe I induces the propagation of promoter melting by directing downstream promoter DNA duplex towards the downstream DNA-binding channel (b 0 clamp). Establishment of downstream contacts leads to remodeling of upstream interactions between s 70 and the K10 promoter element that might facilitate promoter escape and s release.
Methods in enzymology, 2003
The DNA-dependent RNA polymerase (RNAP; EC 2.7.7.6) of Escherichia coli, the best-characterized multisubunit RNAP, is composed of a core enzyme (E, subunit composition 2 0 !) and one of seven identified molecular species of the subunit (E, subunit composition 2 0 !). Advances in high-resolution structural analysis of the bacterial RNAP have opened up opportunities to study the functional role that each structural module of the RNAP plays in transcription. 1,2 A mobile structural module of E. coli RNAP, known as the downstream lobe (residues 186-433), was shown to contribute to stable open promoter complex formation during transcription directed by RNAP containing the 70 factor. 3 This article describes experimental systems used to probe the function of the subunit downstream lobe in the context of RNAP containing the major variant subunit, the enhancer-dependent factor, 54 . 4 Both enhancer-dependent RNAP ( 54 -RNAP) and enhancer-independent RNAP ( 70 -RNAP) are capable of promoter recognition that results in the formation of the closed promoter complex. 70 -RNAP closed promoter complexes can isomerize rapidly into transcriptionally active open promoter complexes in the absence of additional activators or energy sources. In contrast, 54 -RNAP complexes remain closed unless an enhancer DNA-bound activator and an energy source in the form of ATP or GTP hydrolysis is provided. The ATPase activity of the activator induces the propagation of initial DNA melting or distortion in the closed 54 -RNAP promoter complexes toward the transcription initiation start point and allows open promoter complex
Inhibition of a transcriptional pause by RNA anchoring to RNA polymerase
Molecular cell, 2008
We describe a mechanism by which nascent RNA inhibits transcriptional pausing. PutL RNA of bacteriophage HK022 suppresses transcription termination at downstream terminators and pausing within a nearby U-rich sequence. In vitro transcription and footprinting assays reveal that this pausing results from backtracking of RNA polymerase and that binding of nascent putL RNA to polymerase limits backtracking by restricting re-entry of the transcript into the RNA exit channel. The restriction is local and relaxes as the transcript elongates. Our results suggest that putL RNA binds to the surface of polymerase close to the RNA exit channel, a region that includes amino acid residues important for antitermination. Although binding is essential for antipausing and antitermination, these two activities of put differ: antipausing is limited to the immediate vicinity of the putL site, but antitermination is not. We propose that RNA anchoring to the elongation complex is a widespread mechanism of...