Retention of Transcription Initiation Factor ¿ 70 in Transcription Elongation: Single-Molecule Analysis (original) (raw)
Related papers
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 sigma(70) contents of bacterial transcription elongation complexes in vitro. The results confirm ensemble results indicating that a large fraction, similar to 70%-90%, of early elongation complexes retain sigma(70) and that a determinant for sigma(70) recognition in the initial transcribed region increases sigma(70) retention in early elongation complexes. The results establish that a significant fraction, similar to 50%-60%, of mature elongation complexes retain sigma(70) and that a determinant for sigma(70) recognition in the initial transcribed region does not appreciably affect sigma 70 retention in mature elongation complexes. The results further establish that, in mature elongation complexes that retain sigma(70) the half-life of sigma(70) retention is long relative to the timescale of elongation, suggesting that some complexes may retain sigma(70) throughout elongation.
Active Escherichia coli Transcription Elongation Complexes are Functionally Homogeneous
Journal of Molecular Biology, 2002
The elongation phase of RNA transcription represents a major target for the regulation of gene expression. Two general classes of models have been proposed to define the dynamic properties of transcription complexes in the elongation phase. Stable heterogeneity models posit that the ensemble of active elongation-competent complexes consists of multiple distinct and stable forms that are specified early in the transcription cycle and isomerize to other forms slowly. In contrast, equilibrium or rapid interconversion models require that active elongation complexes interconvert rapidly on the time-scale of single nucleotide addition. Measurements of transcription termination efficiency (TE) can be used to distinguish between these models, because stable heterogeneity models predict that the termination-resistant fraction of an elongation complex population should be enriched after transcription through an upstream terminator, leading to a decreased TE at downstream terminators. In contrast, rapid interconversion models require that the population of active (elongation-competent) complexes equilibrate after transcription through each terminator and, therefore, that the value of TE observed at identical upstream and downstream terminators should be the same. We have constructed transcription templates containing multiple identical terminators and found no significant changes in TE with terminator position along the template. Various other forms of upstream treatment of elongation complex populations also were used to attempt to fractionate the complexes into functionally different forms. None of these treatments changed the apparent TE at downstream terminators. These results are consistent with a rapid interconversion model of transcript elongation. The consequences of these results for the regulation of gene expression are discussed.
Bacterial transcription elongation factors: new insights into molecular mechanism of action
Molecular Microbiology, 2004
In this review, we discuss the recent progress in structural and biochemical studies of three evolutionarily conserved elongation factors, GreA, NusA and Mfd. These factors affect RNA polymerase (RNAP) processivity by modulating transcription pausing, arrest, termination or anti-termination. With structural information now available for RNAP and models of ternary elongation complexes, the interaction between these factors and RNAP can be modelled, and possible molecular mechanisms of their action can be inferred. The models suggest that these factors interact with RNAP at or near its three major, nucleic acid-binding channels: Mfd near the upstream opening of the primary (DNA-binding) channel, NusA in the vicinity of both the primary channel and the RNA exit channel, and GreA within the secondary (backtracked RNA-binding) channel, and support the view that these channels are involved in the maintenance of RNAP processivity.
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.
Journal of Molecular Biology, 2011
Initiation of RNA synthesis from DNA templates by RNA polymerase (RNAP) is a multi-step process, in which initial recognition of promoter DNA by RNAP triggers a series of conformational changes in both RNAP and promoter DNA. The bacterial RNAP functions as a molecular isomerization machine, using binding free energy to remodel the initial recognition complex, placing downstream duplex DNA in the active site cleft and then separating the nontemplate and template strands in the region surrounding the start site of RNA synthesis. In this initial unstable "open" complex the template strand appears correctly positioned in the active site. Subsequently, the nontemplate strand is repositioned and a clamp is assembled on duplex DNA downstream of the open region to form the highly stable open complex, RP o . The transcription initiation factor, σ 70 , plays critical roles in promoter recognition and RP o formation as well as in early steps of RNA synthesis.
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.
Molecular Microbiology, 2004
Like transcription initiation, the elongation and termination stages of transcription cycle serve as important targets for regulatory factors in prokaryotic cells. In this review, we discuss the recent progress in structural and biochemical studies of three evolutionarily conserved elongation factors, GreA, NusA and Mfd. These factors affect RNA polymerase (RNAP) processivity by modulating transcription pausing, arrest, termination or anti-termination. With structural information now available for RNAP and models of ternary elongation complexes, the interaction between these factors and RNAP can be modelled, and possible molecular mechanisms of their action can be inferred. The models suggest that these factors interact with RNAP at or near its three major, nucleic acid-binding channels: Mfd near the upstream opening of the primary (DNA-binding) channel, NusA in the vicinity of both the primary channel and the RNA exit channel, and GreA within the secondary (backtracked RNA-binding) channel, and support the view that these channels are involved in the maintenance of RNAP processivity.
2019
ABSTRACTAll organisms--bacteria, archaea, and eukaryotes--have a transcription initiation factor that contains a structural module that binds within the RNA polymerase (RNAP) active-center cleft and interacts with template-strand single-stranded DNA (ssDNA) in the immediate vicinity of the RNAP active center. This transcription-initiation-factor structural module pre-organizes template-strand ssDNA to engage the RNAP active center, thereby facilitating binding of initiating nucleotides and enabling transcription initiation from initiating mononucleotides. However, this transcription-initiation-factor structural module occupies the path of nascent RNA and thus presumably must be displaced before or during initial transcription. Here, we report four sets of crystal structures of bacterial initially transcribing complexes that demonstrate, and define details of, stepwise, RNA-extension-driven displacement of the “σ finger” of the bacterial transcription initiation factor σ. The structu...
Transcription Factor E Is a Part of Transcription Elongation Complexes
Journal of Biological Chemistry, 2007
A homologue of the N-terminal domain of the ␣ subunit of the general eukaryotic transcription factor TFE is encoded in the genomes of all sequenced archaea, but the position of archaeal TFE in transcription complexes has not yet been defined. We show here that TFE binds nonspecifically to single-stranded DNA, and photochemical cross-linking revealed TFE binding to a preformed open transcription bubble. In preinitiation complexes, the N-terminal part of TFE containing a winged helixturn-helix motif is cross-linked specifically to DNA of the nontemplate DNA strand at positions ؊9 and ؊11. In complexes stalled at ؉20, TFE cross-linked specifically to positions ؉9, ؉11, and ؉16 of the non-template strand. Analyses of transcription complexes stalled at position ؉20 revealed a TFE-dependent increase of the resumption efficiency of stalled RNA polymerase and a TFE-induced enhanced permanganate sensitivity of thymine residues in the transcription bubble. These results demonstrate the presence of TFE in early elongation complexes and suggest a role of TFE in stabilization of the transcription bubble during elongation.