Biochemistry. RNA polymerase, a scrunching machine.
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Biomechanical Phenomena↗
Biomedical subjects
Jeffrey W Roberts
Publications and source records attributed to Jeffrey W Roberts.
RNA-mediated destabilization of the sigma(70) region 4/beta flap interaction facilitates engagement of RNA polymerase by the Q antiterminator.
The bacterial RNA polymerase (RNAP) holoenzyme consists of a catalytic core enzyme (alpha(2)betabeta'omega) complexed with a sigma factor that is required for promoter-specific transcription initiation. During early elongation, the stability of interactions between sigma(70) (the primary sigma factor in Escherichia coli) and core decreases due to an ordered displacement of segments of sigma(70) from core triggered by growth of the nascent RNA. Here we demonstrate that the nascent RNA-mediated destabilization of an interaction between sigma(70) region 4 and the flap domain of the beta subunit is required for the bacteriophage lambda Q antiterminator protein to contact holoenzyme during early elongation. We demonstrate further that the requirement for nascent RNA in the process by which Q engages RNAP can be bypassed if sigma(70) region 4 is removed. Our findings illustrate how a regulator can exploit the nascent RNA-mediated reconfiguration of the holoenzyme to gain access to the enzyme during early elongation.
Base Sequence↗
Kinetic investigation of Escherichia coli RNA polymerase mutants that influence nucleotide discrimination and transcription fidelity.
Recent RNA polymerase (RNAP) structures led to a proposed three-step model of nucleoside triphosphate (NTP) binding, discrimination, and incorporation. NTPs are thought to enter through the secondary channel, bind to an E site, rotate into a pre-insertion (PS) site, and ultimately align in the catalytic (A) site. We characterized the kinetics of correct and incorrect incorporation for several Escherichia coli RNAPs with substitutions in the proposed NTP entry pore (secondary channel). Substitutions of the semi-conserved residue betaAsp(675), which is >10A away from these sites, significantly reduce fidelity; however, substitutions of the totally conserved residues betaArg(678) and betaAsp(814) do not significantly alter the correct or incorrect incorporation kinetics, even though the corresponding residues in RNAPII crystal structures appear to be interacting with the NTP phosphate groups and coordinating the second magnesium ion in the active site, respectively. Structural analysis suggests that the lower fidelity of the betaAsp(675) mutants most likely results from reduction of the negative potential of a small pore between the E and PS sites and elimination of several structural interactions around the pore. We suggest a mechanism of nucleotide discrimination that is governed both by rotation of the NTP through this pore and subsequent rearrangement or closure of RNAP to align the NTP in the A site.
Amino Acid Sequence↗
Role of DNA bubble rewinding in enzymatic transcription termination.
By using DNA heteroduplexes that inhibit rewinding of the upstream part of the transcription bubble, we show that transcript release in termination by the enzymes Mfd and Rho is facilitated by reannealing of DNA in the upstream region of the transcription bubble, as is also true for termination by intrinsic terminators. We also show that, like Mfd, the Rho termination factor promotes forward translocation of RNA polymerase. These results support termination models in which external forces imposed on nucleic acids induce concerted rewinding of DNA and unwinding of the DNA/RNA hybrid, possibly accompanied by forward translocation of RNA polymerase, leading to transcription complex dissociation.
Bacterial Proteins↗
An open letter to Elias Zerhouni.
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Biological Warfare↗
A single-molecule technique to study sequence-dependent transcription pausing.
We present a technique that allows sequence-dependent analysis of transcription elongation using single-molecule optical trapping techniques. Observation of individual molecules of RNA polymerase (RNAP) allows determination of elongation kinetics that are difficult or impossible to accurately obtain from bulk studies, and provides high temporal resolution of the RNAP motion under a calibrated mechanical load. One limitation of previous single molecule studies was the difficulty in correlating the observed motion of RNAP with its actual position on the DNA template to better than approximately 100 bp. In this work, we improved the spatial precision of optical trapping studies of transcription to approximately 5 bp by using runoff transcription as an unambiguous marker of RNAP template position. This runoff method was sufficient to unequivocally locate and study a single known pause sequence (DeltatR2). By applying various loads to assist RNAP forward translocation, we specifically investigated elongation kinetics within this pause region and found that the dwell time at the pause sequence decreased with increasing assisting load. This observation is consistent with bulk biochemical studies that suggest RNAP reverse translocates, or "backtracks," at the DeltatR2 pause sequence.
Base Sequence↗
Forward translocation is the natural pathway of RNA release at an intrinsic terminator.
Intrinsic terminators of bacterial RNA polymerase are small (< approximately 30 bp) sequences containing a dyad symmetry that encodes a hairpin in the RNA, followed immediately by a uridine-rich stretch of 5-9 nucleotides just before the site of RNA release. Formation of the RNA hairpin destabilizes the elongation complex, leading to transcript release. We test a model in which hair-pin formation drives RNA polymerase and the melted DNA bubble downstream without transcript elongation, thus releasing the transcript from its enclosure within the enzyme as an RNA/DNA hybrid. We show that blocking downstream translocation of RNAP and preventing downstream DNA unwinding both inhibit transcript release. We argue that translocation of RNA polymerase is essential and that translocation of the bubble stimulates, but is not required, for RNA release; we conclude that forward translocation is the natural pathway of RNA release at an intrinsic terminator.
Bacterial Proteins↗
Interactions among CII protein, RNA polymerase and the lambda PRE promoter: contacts between RNA polymerase and the -35 region of PRE are identical in the presence and absence of CII protein.
The DNA recognition sequence for the transcriptional activator, CII protein, which is critical for lysogenization by bacteriophage lambda, overlaps the -35 region of the P(RE) promoter. Data presented here show that activation by CII does not change the pattern of cleavage of the -35 region of P(RE) by iron (S)-1-(p-bromoacetamidobenzyl)-EDTA (Fe-BABE) conjugated to the sigma subunit of RNA polymerase (RNAP). Thus, the overall interaction between sigma and the -35 region of P(RE) is not significantly altered by CII. Therefore, the effects of the activator on RNAP binding to the promoter and formation of open complexes do not reflect a large-scale qualitative change in the nature of the interaction between RNAP and promoter DNA. The ability of CII to stimulate lysogenization is reduced in the presence of plasmid-borne rpoA variants encoding alanine substitutions at several positions in the C-terminal domain of the alpha subunit. However, it has not been possible to identify residues that directly affect the interaction between the activator and RNA polymerase.
Amino Acid Substitution↗
DNA binding regions of Q proteins of phages lambda and phi80.
Bacteriophage lambda gene Q protein and the related proteins of other lambdoid phages are transcription antiterminators that interact both with DNA in the late gene promoter segment and with RNA polymerase subunits. Using hybrids between Q of lambda and the related Q of phage 80, we characterized elements of both Q and DNA that contribute to the DNA binding function. In particular, we found a C-terminal segment of the protein that is responsible for binding specificity and an approximately 15 residue segment on a predicted alpha helix within this segment at which alanine substitutions decrease DNA binding. We identified a six-nucleotide segment located between the -35 and -10 promoter elements that confers binding specificity and is the site of point mutants that impair binding, and we isolated suppressors in lambda Q that restore binding function by increasing the overall binding affinity. We also identified putative zinc finger structures in both proteins.
Amino Acid Sequence↗
Role of the non-template strand of the elongation bubble in intrinsic transcription termination.
Intrinsic transcription terminators of Escherichia coli and other bacteria, consisting primarily of an RNA hairpin preceding a terminal uridine-rich RNA segment, suffice to dissociate the otherwise stable elongation complex of core RNA polymerase. The essential functions of the hairpin and U-rich segments have been established, although the precise mechanism of termination is unknown. We identify another element of the terminator, namely the non-template DNA strand in the region of the terminal transcription bubble. Failure of the terminal bubble to rewind through complementary base-pairing strongly reduces the efficiency of terminator function, suggesting that the natural pathway of termination consists of coupled rewinding of the DNA template and unwinding of the RNA/DNA hybrid at the site of release.
Base Sequence↗
RNA polymerase mutations that impair conversion to a termination-resistant complex by Q antiterminator proteins.
Bacteriophage lambda Q-protein stably binds and modifies RNA polymerase (RNAP) to a termination-resistant form. We describe amino acid substitutions in RNAP that disrupt Q-mediated antitermination in vivo and in vitro. The positions of these substitutions in the modeled RNAP/DNA/RNA ternary elongation complex, and their biochemical properties, suggest that they do not define a binding site for Q in RNAP, but instead act by impairing interactions among core RNAP subunits and nucleic acids that are essential for Q modification. A specific conjecture is that Q modification stabilizes interactions of RNAP with the DNA/RNA hybrid and optimizes alignment of the nucleic acids in the catalytic site. Such changes would inhibit the activity of the RNA hairpin of an intrinsic terminator to disrupt the 5'-terminal bases of the hybrid and remove the RNA 3' terminus from the active site.
Bacterial Proteins↗
Formation of long DNA templates containing site-specific alkane-disulfide DNA interstrand cross-links for use in transcription reactions.
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Alkanes↗
Single molecule analysis of RNA polymerase elongation reveals uniform kinetic behavior.
By using single-molecule measurements, we demonstrate that the elongation kinetics of individual Escherichia coli RNA polymerase molecules are remarkably homogeneous. We find no evidence of distinct elongation states among RNA polymerases. Instead, the observed heterogeneity in transcription rates results from statistical variation in the frequency and duration of pausing. When transcribing a gene without strong pause sites, RNA polymerase molecules display transient pauses that are distributed randomly in both time and distance. Transitions between the active elongation mode and the paused state are instantaneous within the resolution of our measurements (<1 s). This elongation behavior is compared with that of a mutant RNA polymerase that pauses more frequently and elongates more slowly than wild type.
DNA-Directed RNA Polymerases↗
E. coli Transcription repair coupling factor (Mfd protein) rescues arrested complexes by promoting forward translocation.
Transcription and DNA repair are coupled in E. coli by the Mfd protein, which dissociates transcription elongation complexes blocked at nonpairing lesions and mediates recruitment of DNA repair proteins. We show that Mfd influences the elongation state of RNA polymerase (RNAP); transcription complexes that have reverse translocated into the backtracked position, a potentially important intermediate in RNA proofreading and repair, are restored to the forward position by the activity of Mfd, and arrested complexes are rescued into productive elongation. Mfd may act through a translocase activity that rewinds upstream DNA, leading either to translocation or to release of RNA polymerase when the enzyme active site cannot continue elongation.
Bacterial Proteins↗
RfaH, a bacterial transcription antiterminator.
The bacterial transcription antiterminator RfaH has been shown to act, in a purified biochemical system, by binding both RNA polymerase and the nontemplate strand of DNA at the regulatory site ops.
DNA, Bacterial↗
The sigma(70) subunit of RNA polymerase is contacted by the (lambda)Q antiterminator during early elongation.
The Q protein of bacteriophage lambda is a transcription antiterminator that modifies the elongation properties of E. coli RNA polymerase (RNAP). To do this, DNA-bound (lambda)Q must first engage a paused elongation complex. Here we show that this engagement of (lambda)Q with RNAP involves an interaction between (lambda)Q and sigma(70), demonstrating that sigma(70) can be a target of regulation during elongation. Furthermore, we provide evidence that this interaction between (lambda)Q and sigma(70) stabilizes a conformation of RNAP that requires the disengagement of a segment of sigma(70) from the core enzyme. Recent structure-based models posit that the transition from the initiation to the elongation phase of transcription involves the staged displacement of sigma(70) from the RNAP core. Our findings provide support for this proposal.
Bacteriophage lambda↗