Jeffrey W. Roberts

Nature of the SOS-inducing signal in Escherichia coli: The involvement of DNA replication☆

The SOS genes of Escherichia coli, which include many DNA repair genes, are induced by DNA damage. Although the central biochemical event in induction, activation of RecA protein through binding of single-stranded DNA and ATP to promote cleavage of the LexA repressor, is known, the cellular event that provides this activation following DNA damage has not been well understood. We provide evidence here that the major pathway of induction after damage by a typical agent, ultraviolet light, requires an active replication fork; this result supports the model that DNA replication leaves gaps where elongation stops at damage-induced lesions, and thus provides the single-stranded DNA that activates RecA protein. In order to detect quantitatively the immediate product of the inducing signal, activated RecA protein, we have designed an assay to measure the rate of disappearance of intact LexA repressor. With this assay, we have studied the early phase of the induction process. LexA cleavage is detectable within minutes after DNA damage and occurs in the absence of protein synthesis. By following the reaccumulation of LexA in the cell, we detect repair of DNA and the disappearance of the inducing signal. Using this assay, we have measured the LexA content of wild-type and various mutant cells, characterized the kinetics and conditions for development of the inducing signal after various inducing treatments and, finally, have shown the requirement for DNA replication in SOS induction by ultraviolet light.

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.

Function of E. coli RNA Polymerase σ Factor- σ70 in Promoter-Proximal Pausing

The sigma factor sigma 70 of E. coli RNA polymerase acts not only in initiation, but also at an early stage of elongation to induce a transcription pause, and simultaneously to allow the phage lambda gene Q transcription antiterminator to act. We identify the signal in DNA that induces early pausing to be a version of the sigma 70 -10 promoter consensus, and we show that sigma 70 is both necessary for pausing and present in the paused transcription complex. Regions 2 and 3 of sigma 70 suffice to induce pausing. Since pausing is induced by the nontemplate DNA strand of the open transcription bubble, we conclude that RNA polymerase containing sigma 70 carries out base-specific recognition of the nontemplate strand as single stranded DNA. We suggest that sigma 70 remains bound to core RNA polymerase when the -10 promoter contacts are broken, and then moves to the pause-inducing sequence.

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.

Induction of SOS functions: Regulation of proteolytic activity of E. coil RecA protein by interaction with DNA and nucleoside triphosphate

Damage to cellular DNA or interruption of chromosomal DNA synthesis leads to induction of the SOS functions in E. coli. The immediate agent of induction is the RecA protein, which proteolytically cleaves and inactivates repressors, leading to induction of genes they control. RecA protein modified by tif mutations allows expression of SOS functions in the absence of inducing treatments. We show here that tif-mutant RecA protein is more efficient than wild-type RecA protein in interacting with DNA and nucleoside triphosphate. This result suggests that formation of a complex with DNA and nucleoside triphosphate is the critical event that activates RecA protein to destroy repressors after SOS-inducing treatments, and that damage to cellular DNA promotes this reaction by providing single-stranded DNA or active nucleoside triphosphate or both. Since dATP is the most effective nucleoside triphosphate in promoting repressor cleavage, we suggest that it is the natural cofactor of recA protein in vivo.

Base-Specific Recognition of the Nontemplate Strand of Promoter DNA by E. coli RNA Polymerase

RNA polymerase recognizes its promoters through base-specific interaction between defined segments of DNA and the sigma subunit of the enzyme. This interaction leads to separation of base pairs and exposure of the template strand for RNA synthesis. We show that base-specific recognition by the sigma 70 holoenzyme in this process involves primarily nontemplate strand bases in the -10 promoter region. We suggest that melting involves the persistence of these contacts as the bound duplex (closed) form is converted to the single-stranded (open) form of the enzyme-promoter complex.

Function of Transcription Cleavage Factors GreA and GreB at a Regulatory Pause Site

Gre proteins of prokaryotes, and SII proteins of eukaryotes and archaea, are transcription elongation factors that promote an endogenous transcript cleavage activity of RNA polymerases; this process promotes elongation through obstructive regions of DNA, including transcription pauses that act as sites of genetic regulation. We show that a regulatory pause in the early part of the late gene operon of bacteriophage lambda is subject to such cleavage and resynthesis. In cells lacking the cleavage factors GreA and GreB, the pause is prolonged, and RNA polymerase occupies a variant position at the pause site. Furthermore, GreA and GreB are required to mediate efficient function of the lambda gene Q antiterminator at this site. Thus, cleavage factors are necessary for the natural progression of RNA polymerase in vivo.

Phage lambda gene Q antiterminator recognizes RNA polymerase near the promoter and accelerates it through a pause site

The positive regulator encoded by phage lambda gene Q is a transcription antiterminator that affects RNA polymerase initiating at the phage late gene promoter, but not at other promoters. We show that this nucleotide-sequence-specific interaction of Q protein and RNA polymerase can occur while the enzyme is pausing after 16 nucleotides of the late gene transcript have been made. Furthermore, Q protein chases RNA polymerase from this early pause site, so that it both recognizes the enzyme and changes its transcription properties at this site. We suggest that the ability of Q-modified RNA polymerase to escape this pause reflects the change that allows it to go through terminators. We also show that NusA protein is required for efficient Q protein activity in vitro.

RNA Polymerase Elongation Factors

The elongation phase of transcription by RNA polymerase is highly regulated and modulated. Both general and operon-specific elongation factors determine the local rate and extent of transcription to coordinate the appearance of transcript with its use as a messenger or functional ribonucleoprotein or regulatory element, as well as to provide operon-specific gene regulation.

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.