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Specific small deletions within the rpoC gene encoding the [beta]-subunit of RNA polymerase (RNAP) are found repeatedly after adaptation of Escherichia coli K-12 MG1655 to growth in minimal media. Here we present a multiscale analysis of these mutations. At the physiological level, the mutants grow 60% faster than the parent strain and convert the carbon source 15-35% more efficiently to biomass, but grow about 30% slower than the parent strain in rich medium. At the molecular level, the kinetic parameters of the mutated RNAP were found to be altered, resulting in a 4- to 30-fold decrease in open complex longevity at an rRNA promoter and a ~ 10-fold decrease in transcriptional pausing, with consequent increase in transcript elongation rate. At a genome-scale, systems biology level, gene expression changes between the parent strain and adapted RNAP mutants reveal large-scale systematic transcriptional changes that influence specific cellular processes, including strong down-regulation of motility, acid resistance, fimbria, and curlin genes. RNAP genome-binding maps reveal redistribution of RNAP that may facilitate relief of a metabolic bottleneck to growth. These findings suggest that reprogramming the kinetic parameters of RNAP through specific mutations allows regulatory adaptation for optimal growth in new environments. kinetics | stringent response | transcription doi/ 10.1073/pnas.0911253107

We report observations suggesting that the transcription elongation factor NusA promotes a previously unrecognized class of transcription-coupled repair (TCR) in addition to its previously proposed role in recruiting translesion synthesis (TLS) DNA polymerases to gaps encountered during transcription. Earlier, we reported that NusA physically and genetically interacts with the TLS DNA polymerase DinB (DNA pol IV). We find that Escherichia coil nusA11(ts) mutant strains, at the permissive temperature, are highly sensitive to nitrofurazone (NFZ) and 4-nitroquinolone-1-oxide but not to UV radiation. Gene expression profiling suggests that this sensitivity is unlikely to be due to an indirect effect on gene expression affecting a known DNA repair or damage tolerance pathway. We demonstrate that an [N.sup.2]-furfuryl-dG ([N.sup.2]-f-dG) lesion, a structural analog of the principal lesion generated by NFZ, blocks transcription by E. coli RNA polymerase (RNAP) when present in the transcribed strand, but not when present in the nontranscribed strand. Our genetic analysis suggests that NusA participates in a nucleotide excision repair (NER)-dependent process to promote NFZ resistance. We provide evidence that transcription plays a role in the repair of NFZ-induced lesions through the isolation of RNAP mutants that display altered ability to survive NFZ exposure. We propose that NusA participates in an alternative class of TCR involved in the identification and removal of a class of lesion, such as the [N.sup.2]-f-dG lesion, which are accurately and efficiently bypassed by DinB in addition to recruiting DinB for TLS at gaps encountered by RNAP. excision repair | RNA polymerase | translesion synthesis | Mfd | transcription-coupled doi/ 10.1073/pnas.1005203107

The transcription termination factor Rho is a global regulator of RNA polymerase (RNAP). Although individual Rho-dependent terminators have been studied extensively, less is known about the sites of RNAP regulation by Rho on a genome-wide scale. Using chromatin immunoprecipitation and microarrays (ChIP-chip), we examined changes in the distribution of Escherichia coil RNAP in response to the Rho-specific inhibitor bicyclomycin (BCM). We found [approximately equal to]200 Rho-terminated loci that were divided evenly into 2 classes: intergenic (at the ends of genes) and intragenic (within genes). The intergenic class contained noncoding RNAs such as small RNAs (sRNAs) and transfer RNAs (tRNAs), establishing a previously unappreciated role of Rho in termination of stable RNA synthesis. The intragenic class of terminators included a previously uncharacterized set of short antisense transcripts, as judged by a shift in the distribution of RNAP in BCM-treated cells that was opposite to the direction of the corresponding gene. These Rho-terminated antisense transcripts point to a role of non-coding transcription in E. coli gene regulation that may resemble the ubiquitous noncoding transcription recently found to play myriad roles in eukaryotic gene regulation. chromatin immunoprecipitation | RNA polymerase

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2008.01.027 Byline: Matthew H. Larson (1), William J. Greenleaf (2), Robert Landick (4), Steven M. Block (2)(3) Keywords: RNA; DNA; PROTEINS Abstract: Transcription termination by bacterial RNA polymerase (RNAP) occurs at sequences coding for a GC-rich RNA hairpin followed by a U-rich tract. We used single-molecule techniques to investigate the mechanism by which three representative terminators (his, t500, and tR2) destabilize the elongation complex (EC). For his and tR2 terminators, loads exerted to bias translocation did not affect termination efficiency (TE). However, the force-dependent kinetics of release and the force-dependent TE of a mutant imply a forward translocation mechanism for the t500 terminator. Tension on isolated U-tracts induced transcript release in a manner consistent with RNA:DNA hybrid shearing. We deduce that different mechanisms, involving hypertranslocation or shearing, operate at terminators with different U-tracts. Tension applied to RNA at terminators suggests that closure of the final 2-3 hairpin bases destabilizes the hybrid and that competing RNA structures modulate TE. We propose a quantitative, energetic model that predicts the behavior for these terminators and mutant variants. Author Affiliation: (1) Biophysics Program, Stanford University, Stanford, CA 94305, USA (2) Department of Applied Physics, Stanford University, Stanford, CA 94305, USA (3) Department of Biological Sciences, Stanford University, Stanford, CA 94305, USA (4) Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA Article History: Received 1 November 2007; Revised 14 December 2007; Accepted 11 January 2008 Article Note: (miscellaneous) Published: March 20, 2008

The architecture of cellular RNA polymerases (RNAPs) dictates that transcription can begin only after promoter DNA bends into a deep channel and the start site nucleotide (+1) binds in the active site located on the channel floor. Formation of this transcriptionally competent 'open' complex ([RP.sub.o]) by Escherichia coli RNAP at the [lambda][P.sub.R] promoter is greatly accelerated by DNA upstream of base pair -47 (with respect to +1). Here we report real-time hydroxyl radical (*OH) and potassium permanganate (KMn[O.sub.4]) footprints obtained under conditions selected for optimal characterization of the first kinetically significant intermediate ([I.sub.1]) in [RP.sub.o] formation. *OH footprints reveal that the DNA backbone from -71 to -81 is engulfed by RNAP in [I.sub.1] but not in [RP.sub.o]; downstream protection extends to approximately +20 in both complexes. KMn[O.sub.4] footprinting detects solvent-accessible thymine bases in [RP.sub.o], but not in [I.sub.1]. We conclude that upstream DNA wraps more extensively on RNAP in [I.sub.1] than in [RP.sub.o] and that downstream DNA (-11 to +20) occupies the active-site channel in [I.sub.1] but is not yet melted. Mapping of the footprinting data onto available x-ray structures provides a detailed model of a kinetic intermediate in bacterial transcription initiation and suggests how transient contacts with upstream DNA in [I.sub.1] might rearrange the channel to favor entry of downstream duplex DNA. initiation | transcription | wrapping | kinetics

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2006.11.036 Byline: Robert Landick (1) Abstract: The 2006 Nobel Prize in Chemistry has been awarded to Roger Kornberg for elucidating the molecular basis of eukaryotic transcription. The prize caps a decades-long quest to unlock one of the central mysteries of molecular biology -- how RNA transcripts are assembled. Author Affiliation: (1) Department of Bacteriology, 420 Henry Mall, University of Wisconsin-Madison, Madison, WI 53706, USA

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2006.04.032 Byline: Kristina M. Herbert (1), Arthur La Porta (2), Becky J. Wong (2), Rachel A. Mooney (3), Keir C. Neuman (2), Robert Landick (3), Steven M. Block (2)(4) Abstract: Transcriptional pausing by RNA polymerase (RNAP) plays an important role in the regulation of gene expression. Defined, sequence-specific pause sites have been identified biochemically. Single-molecule studies have also shown that bacterial RNAP pauses frequently during transcriptional elongation, but the relationship of these 'ubiquitous' pauses to the underlying DNA sequence has been uncertain. We employed an ultrastable optical-trapping assay to follow the motion of individual molecules of RNAP transcribing templates engineered with repeated sequences carrying imbedded, sequence-specific pause sites of known regulatory function. Both the known and ubiquitous pauses appeared at reproducible locations, identified with base-pair accuracy. Ubiquitous pauses were associated with DNA sequences that show similarities to regulatory pause sequences. Data obtained for the lifetimes and efficiencies of pauses support a model where the transition to pausing branches off of the normal elongation pathway and is mediated by a common elemental state, which corresponds to the ubiquitous pause. Author Affiliation: (1) Biophysics Program, Stanford University, Stanford, CA 94305, USA (2) Department of Biological Sciences, Stanford University, Stanford, CA 94305, USA (3) Department of Bacteriology, University of Wisconsin -- Madison, Madison, WI 53706, USA (4) Department of Applied Physics, Stanford University, Stanford, CA 94305, USA Article History: Received 24 January 2006; Revised 18 March 2006; Accepted 13 April 2006 Article Note: (miscellaneous) Published: June 15, 2006

The recent elucidation of crystal structures for multi-subunit RNA polymerases immediately revealed a mystery: how do nucleotide triphosphate (NTP) substrates reach an active site that is buried deep within the enzyme? The prevailing view is that NTPs enter through an ~20A-Iong secondary channel between the active site and the enzyme surface. Recently, an alternative view has been advocated; namely, NTPs enter the active site pre-bound to the DNA template from the downstream DNA portion of the main channel of the enzyme.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.tibs.2005.10.001 Byline: Robert Landick Abstract: The recent elucidation of crystal structures for multi-subunit RNA polymerases immediately revealed a mystery: how do nucleotide triphosphate (NTP) substrates reach an active site that is buried deep within the enzyme? The prevailing view is that NTPs enter through an [approximately equal to]20a'-long secondary channel between the active site and the enzyme surface. Recently, an alternative view has been advocated; namely, NTPs enter the active site pre-bound to the DNA template from the downstream DNA portion of the main channel of the enzyme. Author Affiliation: Department of Bacteriology, University of Wisconsin-Madison, Madison, WI 53706, USA

Formation of productive transcription complexes after promoter escape by RNA polymerase II is a major event in eukaryotic gene regulation. Both negative and positive factors control this step. The principal negative elongation factor (NELF) contains four polypeptides and requires for activity the two-polypeptide 5,6-dichloro-1-[beta]-D-ribobenzimidazole-sensitivity inducing factor (DSIF). DSIF/ NELF inhibits early transcript elongation until it is counteracted by the positive elongation factor P-TEFb. We report a previously undescribed activity of DSIF/NELF, namely inhibition of the transcript cleavage factor TFIIS. These two activities of DSIF/NELF appear to be mechanistically distinct. Inhibition of nucleotide addition requires [greater than or equal to] 18 nt of nascent RNA, whereas inhibition of TFIIS occurs at all transcript lengths. Because TFIIS promotes escape from promoter-proximal pauses by stimulating cleavage of backtracked nascent RNA, TFIIS inhibition may help DSIF/NELF negatively regulate productive transcription. transcription elongation | pausing | backtracking

The genome-wide location of RNA polymerase binding sites was determined in Escherichia coli using chromatin immunoprecipitation and microarrays (chIP-chip). Cross-linked chromatin was isolated in triplicate from rifampin-treated cells, and DNA bound to RNA polymerase was precipitated with an antibody specific for the [beta]' subunit. The DNA was amplified and hybridized to 'tiled' oligonucleotide microarrays representing the whole genome at 25-bp resolution. A total of 1,139 binding sites were detected and evaluated by comparison to gene expression data from identical conditions and to 961 promoters previously identified by established methods. Of the detected binding sites, 418 were located within 1,000 bp of a known promoter, leaving 721 previously unknown RNA polymerase binding sites. Within 200 bp, we were able to detect 51% (189/368) of the known [sigma]70-specific promoters occurring upstream of an expressed open reading frame and 74% (273/368) within 1,000 bp. Conversely, many known promoters were not detected by chIP-chip, leading to an estimated 26% negative-detection rate. Most of the detected binding sites could be associated with expressed transcription units, but 299 binding sites occurred near inactive transcription units. This map of RNA polymerase binding sites represents a foundation for studies of transcription factors in E. coli and an important evaluation of the chIP-chip technique.

Author(s): Joshua W. Shaevitz [1, 5]; Elio A. Abbondanzieri [2, 5]; Robert Landick [4]; Steven M. Block (corresponding author) [2, 3] Escherichia coli RNA polymerase (RNAP) synthesizes RNA with remarkable [...]

Bacterial RNAPs typically consist of five polypeptides: β, 'β, α, and w. β' and β form a main channel that holds the RNA 3' OH in the active site, an [...], RNA polymerase (RNAP) is the central enzyme of gene expression. Despite availability of crystal structures, details of its nucleotide addition cycle remain obscure. We describe bacterial RNAP inhibitors (the CBR703 series) whose properties illuminate this mechanism. These compounds inhibit known catalytic activities of RNAP (nucleotide addition, pyrophosphorolysis, and Gre-stimulated transcript cleavage) but not translocation of RNA or DNA when translocation is uncoupled from catalysis. CBR703-resistance substitutions occur on an outside surface of RNAP opposite its internal active site. We propose that CBR703 compounds inhibit nucleotide addition allosterically by hindering movements of active site structures that are linked to the CBR703 binding site through a bridge helix.

Efficient synthesis of long messenger RNAs encounters difficulties during gene expression due to pausing, arrest, and termination activities of transcription complex structures. Research reveals that transcription regulation is mediated by elongation/antitermination factors stabilizing RNA polymerase-nucleic acid interactions, that allows RNA polymerase to ignore pausing, arrest, and termination signals.

Results demonstrate that the RNA polymerases from Bacillus subtilis and Escherichia coli exhibit both common and species-specific differences in their response to various regulatory signals. Data reveal that while both respond similarly to pause and termination signals, promoter discrimination and signal recognition responses are species-specific.

Multisubunit RNA polymerases (RNAPs), which synthesize essentially all cellular RNA molecules, are discussed. Their core subunits are conserved all through evolution. A paradigm of interactions of core RNAP with nucleic acid and accessory factors driving its passage through a series of structurally distinct intermediates in a transcription cycle is based on decades of research. New models of the transcription elongation complex (TEC) have emerged and are largely consistent with the previous paradigm, suggesting that conversion to the TEC depends on closure of parts of RNAP around the RNA:DNA hybrid and the nascent RNA on its way out, a closed conformation.

We compared in vitro transcription-initiated folding of the ribozyme from Bacillus subtilis RNase P to refolding from the full-length, denatured state by monitoring the appearance of its catalytic activity. At 37 [degrees] C, [Mg.sup.2+]-initiated refolding of the wild type and a circularly permutate ribozyme takes minutes and is limited by a kinetic trap. Transcription by T7 RNA polymerase alters the folding pathway of both RNAs and introduces new kinetic traps. Transcription by the core Escherichia coli RNA polymerase yields the same result, in spite of its 4-fold-slower elongation rate. However, the presence of its elongation factor NusA accelerates more than 10-fold the transcription-initiated folding of the circularly, permutated ribozyme by E. coli RNA polymerase. The effect of NusA likely is caused by its enhancement of transcriptional pausing because NusA did not accelerate transcription-initiated folding using a mutant RNA polymerase that failed to pause or respond to NusA during ribozyme synthesis. We conclude that both transcription and specific pausing therein can alter RNA-folding pathways.

Nascent RNA structures may exert control over elongation or RNA chains directly by interacting with RNA polymerase or in an indirect way by disrupting RNA contacts at the nascent stage with DNA or polymerase. Effects of the his (ital) leader pause RNA hairpin have been tested for ability to be mimicked by pairing of antisense DNA or RNA oligonucleotides to the nascent transcript. It was shown that a nascent RNA structure must interact with RNA polymerase for hairpin-dependent transcriptional pausing. It is not necesary for transcript release.

A review was done to describe the mechanical processes of the RNA polymerase (RNAP), with particular interest on the movement of single RNAP molecules along the DNA. Cellular RNAPs are homologous multisubunit enzymes that executive a series of coordinated movements while transcribing DNA. Single-molecule techniques for analyzing RNAP translocation along the DNA, showed that the enzyme has the ability to translocate DNA against large opposing loads and that deduced efficiency of chemical-to-mechanical energy conversion is high which is similar to canonical cytoskeletal motor enzymes.

RNA secondary structures (hairpins) that form as the nascent RNA emerges from RNA polymerase are important components of many signals that regulate transcription, including some pause sites, all [Rho]-independent terminators, and some antiterminators. At the his leader pause site, a 5-bp-stem, 8-nt-loop pause RNA hairpin forms 11 nt from the RNA 3[prime] end and stabilizes a transcription complex conformation slow to react with NTP substrate. This stabilization appears to depend at least in part on an interaction with RNA polymerase. We tested for RNA hairpin interaction with the paused polymerase by crosslinking 5-iodoUMP positioned specifically in the hairpin loop. In the paused conformation, strong and unusual crosslinking of the pause hairpin to [Beta]904-950 replaced crosslinking to [Beta][prime] and to other parts of [Beta] that occurred in nonpaused complexes prior to hairpin formation. These changes in nascent RNA interactions may inhibit reactive alignment of the RNA 3[prime] end in the paused complex and be related to events at [Rho]-independent terminators.

RNA polymerase (RNAP) have been determined to perform the function of facilitating chain elongation as well as recognizing pause signals and termination sites during the process. A sliding-clamp model of the transcription complex has been proposed to explain the mechanism by which RNAP performs such functions. In the model, RNAP encircles duplex DNA ahead of the active site then slides backwards to increase the stability of a constant-sized RNA:DNA hybrid of about 8 bp.

Defective ribonucleic acid (RNA) polymerases of Escherichia coli are isolated by utilizing GalK expression vectors pES4 and pPLT7. The mutant RNA polymerases were unable to support cell growth due to severe deficiencies in RNA polymerase functions. The mutations of the plasmid with deficient RNA polymerases were located on the highly conserved region of the beta subunit of prokaryotic RNA polymerase. The plasmid-encoded mutant beta subunits were unable to restore growth at high temperatures and affect the normal growth in the presence of wild-type RNA polymerases.

RNA polymerases play a critical role in gene expression by synthesizing RNA transcripts containing genetic information copied from DNA templates. The nascent RNA chain is elongated in a chemical reaction [...], The force produced by a single molecule of Escherichia coli RNA polymerase during transcription was measured optically. Polymerase immobilized on a surface was used to transcribe a DNA template attached to a polystyrene bead 0.5 micrometer in diameter. The bead position was measured by interferometry while a force opposing translocation of the polymerase along the DNA was applied with an optical trap. At saturating nucleoside triphosphate concentrations, polymerase molecules stalled reversibly at a mean applied force estimated to be 14 piconewtons. This force is substantially larger than those measured for the cytoskeletal motors kinesin and myosin and exceeds mechanical loads that are estimated to oppose transcriptional elongation in vivo. The data are consistent with efficient conversion of the free energy liberated by RNA synthesis into mechanical work.

The beta subunit of RNA polymerase (RNAP) appears to be composed of at least three independent domains. Beta subunit fragments encoded by rpoB segments, on combination with purified recombinant beta', sigma, and alpha subunit of RNA, reconstitute a functional enzyme in vitro. These study findings present new ways of investigating RNAP assembly and structure.

Transcriptional pausing could be a result of discontinuous movements of RNA polymerase along DNA and RNA chains. Changes in 3'-proximal or a downward DNA affect discontinuity of DNA and RNA movements accompanying RNA polymerase and pause RNA hairpin restricts the new downward DNA in the paused complex. It is possible that the discontinuous movements regulate both termination and antitermination because stopping the discontinuous movement causes antitermination.

A structural basis for inhibition of bacterial RNA polymerase by the antibiotic streptolydigin is described. Streptolydigin prevents the distortion of a bridge (alpha) helix postulated to occur at the time of nucleotide addition cycle of RNA polymerase or block a small movement of the bridge helix that helps load nucleotide triphosphates into active site.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2005.08.008 Byline: Scotty Kyzer (1), Jinwei Zhang (1), Robert Landick (2) Abstract: Bacterial RNA polymerase is a common target for many antibiotics. In two recent papers in Cell and Molecular Cell, and describe a structural basis for inhibition of bacterial RNA polymerase by the antibiotic streptolydigin. Streptolydigin may prevent distortion of a 'bridge' [alpha] helix postulated to occur during the nucleotide addition cycle of RNA polymerase or may block a small movement of the bridge helix that helps load nucleotide triphosphates into the active site. Author Affiliation: (1) Department of Biomolecular Chemistry, University of Wisconsin, Madison, Wisconsin 53706 (2) Department of Bacteriology, University of Wisconsin, Madison, Wisconsin 53706

Author(s): Dmitry G. Vassylyev (corresponding author) [1]; Marina N. Vassylyeva [1]; Jinwei Zhang [2]; Murali Palangat [3]; Irina Artsimovitch [5]; Robert Landick [3, 4] The mechanism of substrate selection and [...]

An ultrastable optical-trapping assay is developed to follow the motion of individual molecules of ribonucleic acid polymerase (RNAP) transcribing templates engineered with repeated sequences carrying imbedded, sequence-specific pause sites of known regulatory function. The data obtained for the lifetimes and efficiencies of pauses support a model, where the transition to pausing branches off of the normal elongation pathway and is mediated by a common elemental state, which corresponds to the ubiquitous pause.

Author(s): Elio A. Abbondanzieri [1, 6]; William J. Greenleaf [1, 6]; Joshua W. Shaevitz [2, 5]; Robert Landick [4]; Steven M. Block (corresponding author) [1, 3] Processive molecular motors tend [...]

The effective concentration of sigma(super 70) in vivo, and the extent to which can regulate transcript is unknown. A study was conducted which reflected that tethering sigma(super 70) yields viable Escherichia coli strains in which alternative s-factor function is not impaired.

Research reveal that majority of the transcriptional pauses are brief lasting 1-6 seconds at 21 centigrade with less likely of pausing any specific position on a DNA template. Data point out that these transcrptional pauses are due to structural rearrangement within the enzyme.

Research has been conducted on the structure of amino acid peptide inhibitor, microcin J25. Via the use of biochemical studies, NMR and mass spectrometry the authors have demonstrated that this peptide has unusual structural fold.

Research has been conducted on bacteriophage lambda Q-protein which binds RNA polymerase to a termination-resistant form. The authors have investigated amino acid substitutions in this polymerase which disrupts Q-mediate antitermination, and discuss the results.

Evolutionarily conserved, multisubunit RNAPs transcribe genes into RNAs in all known organisms. A universal feature of these RNAPs is a regulated conversion from an initiating form that holds the RNA [...], DNA, RNA, and regulatory molecules control gene expression through interactions with RNA potymerase (RNAP). We show that a short [Alpha] helix at the tip of the flaplike domain that covers the RNA exit channel of RNAP contacts a nascent RNA stem-loop structure (hairpin) that inhibits transcription, and that this flap-tip helix is required for activity of the regulatory protein NusA. Protein-RNA cross-linking, molecular modeling, and effects of alterations in RNAP and RNA all suggest that a tripartite interaction of RNAP, NusA, and the hairpin inhibits nucleotide addition in the active site, which is located 65 angstroms away. These findings favor an attosteric model for regulation of transcript elongation.

Transcript elongation by RNA polymerase is discontinuous and interrupted by pauses that play key regulatory roles. We show here that two different classes of pause signals punctuate elongation. Class I pauses, discovered in enteric bacteria, depend on interaction of a nascent RNA structure with RNA polymerase to displace the 3' OH away from the catalytic center. Class II pauses, which may predominate in eukaryotes, cause RNA polymerase to slide backwards along DNA and RNA and to occlude the active site with nascent RNA. These pauses differ in their responses to antisense oligonucleotides, pyrophosphate, GreA, and general elongation factors NusA and NusG. In contrast, substitutions in RNA polymerase that increase or decrease the rate of RNA synthesis affect both pause classes similarly. We propose that both pause classes, as well as arrest and termination, arise from a common intermediate that itself binds NTP substrate weakly. transcriptional pausing | NUSA | NUSG

Transcription elongation is a key step in the regulation of gene expression in cells. During elongation, RNA polymerase (RNAP) is known to move discontinuously, spending proportionally more time at some [...], Using an optical-trap/flow-control video microscopy technique, we followed transcription by single molecules of Escherichia coil RNA polymerase in real time over long template distances. These studies reveal that RNA polymerase molecules possess different intrinsic transcription rates and different propensities to pause and stop. The data also show that reversible pausing is a kinetic intermediate between normal elongation and the arrested state. The conformational metastability of RNA polymerase revealed by this single-molecule study of transcription has direct implications for the mechanisms of gene regulation in both bacteria and eukaryotes.

To identify the location of a domain of the [Beta]-subunit of Escherichia coli RNA polymerase (RNAP) on the three-dimensional structure, we developed a method to tag a nonessential surface of the multisubunit enzyme with a protein density easily detectable by electron microscopy and image processing. Four repeats of the IgG-binding domain of Staphylococcus aureus protein A were inserted at position 998 of the E. coli RNAP [Beta]-subunit. The mutant RNAP supported E. coli growth and showed no apparent functional defects in vitro. The structure of the mutant RNAP was determined by cryoelectron microscopy and image processing of frozen-hydrated helical crystals. Comparison of the mutant RNAP structure with the previously determined wild-type RNAP structure by Fourier difference analysis at 20-A resolution directly revealed the location of the inserted protein domain, thereby locating the region around position 998 of the [Beta]-subunit within the RNAP three-dimensional structure and refining a model for the subunit locations within the enzyme.

Cessation of transcription at specific terminator DNA sequences is used by viruses, bacteria, and eukaryotes to regulate the expression of downstream genes, but the mechanisms of transcription termination are poorly characterized. To elucidate the kinetic mechanism of termination at the intrinsic terminators of enteric bacteria, we observed, by using single-molecule light microscopy techniques, the behavior of surface-immobilized Escherichia coli RNA polymerase (RNAP) molecules in vitro. An RNAP molecule remains at a canonical intrinsic terminator for [approximately equals] 64 s before releasing DNA, implying the formation of an elongation-incompetent (paused) intermediate by transcription complexes that terminate but not by those that read through the terminator. Analysis of pause lifetimes establishes a complete minimal mechanism of termination in which paused intermediate formation is both necessary and sufficient to induce release of RNAP at the terminator. The data suggest that intrinsic terminators function by a nonequilibrium process in which terminator effectiveness is determined by the relative rates of nucleotide addition and paused state entry by the transcription complex.

THE NIH PEER-REVIEW PROCESS AND NIH investments in research on microbial physiology, genetics, and pathogenesis have made possible remarkable advances in science and public health and have underpinned the development [...]

RNA polymerase synthesizes messenger RNA from the gene. As promised by the early visionaries of molecular biology, regulatory factors that act on RNA polymerase mediate key events in biological development [...]

To direct expression of a gene, RNA polymerase (RNAP) must efficiently transcribe (copy) DNA into an mRNA molecule that is thousands to millions of nucleotides long. Organisms from bacteria to [...], To direct expression of a gene, RNA polymerase (RNAP) must efficiently transcribe (copy) DNA into an mRNA molecule that is thousands to millions of nucleotides long. Organisms from bacteria to humans regulate gene expression by making RNAPs that readily hesitate, stop, or fall off the DNA template when they encounter certain sequences (called pause, arrest, or termination signals). There also exist auxiliary proteins that switch RNAP from a state that is susceptible to pausing, arrest, and termination (defined here as hesitant) into a state that is resistant to these signals (defined here as overdrive). In a Research Article on page 611 of this issue, Yarnell and Roberts (1) explain how the bacteriophage Λ protein Q makes the RNAP of Escherichia coli resistant to termination sequences in the DNA. Their study offers important insights not only into bacterial mechanisms of termination and overdrive (called antitermination in bacteria), but also into an intriguing but poorly understood mechanism of human gene regulation in which RNAPII switches between inefficient (hesitant) and efficient (overdrive) modes of mRNA chain elongation.

RNA polymerase (RNAP) carries out an essential step of gene expression, the synthesis of an RNA copy of the template DNA. Each RNA molecule is synthesized in its entirety by […]