Mechanisms of Bacterial Transcription Termination.

Bacterial transcription termination, described mostly for Escherichia coli, occurs in three recognized ways: intrinsic termination, an activity only of the core RNAP enzyme and transcript sequences that encode an RNA hairpin and terminal uridine-rich segment; termination by the enzyme Rho, an ATP-dependent RNA translocase that releases RNA by forcing uncharacterized structural changes in the elongating complex; and Mfd-dependent termination, the activity of an ATP-dependent DNA translocase that is thought to dissociate the elongation complex by exerting torque on a stalled RNAP. Intrinsic termination can be described in terms of the nucleic acid movements in the process, whereas the enzymatic mechanisms have been illuminated importantly by definitive structural and biochemical analysis of their activity.

Transcription factor regulation of RNA polymerase's torque generation capacity.

During transcription, RNA polymerase (RNAP) supercoils DNA as it translocates. The resulting torsional stress in DNA can accumulate and, in the absence of regulatory mechanisms, becomes a barrier to RNAP elongation, causing RNAP stalling, backtracking, and transcriptional arrest. Here we investigate whether and how a transcription factor may regulate both torque-induced RNAP stalling and the torque generation capacity of RNAP.

Mfd Dynamically Regulates Transcription via a Release and Catch-Up Mechanism.

The bacterial Mfd ATPase is increasingly recognized as a general transcription factor that participates in the resolution of transcription conflicts with other processes/roadblocks. This function stems from Mfd's ability to preferentially act on stalled RNA polymerases (RNAPs). However, the mechanism underlying this preference and the subsequent coordination between Mfd and RNAP have remained elusive.

A universal transcription pause sequence is an element of initiation factor σ70-dependent pausing.

The Escherichia coli σ70 initiation factor is required for a post-initiation, promoter-proximal pause essential for regulation of lambdoid phage late gene expression; potentially, σ70 acts at other sites during transcription elongation as well. The pause is induced by σ70 binding to a repeat of the promoter -10 sequence. After σ70 binding, further RNA synthesis occurs as DNA is drawn (or 'scrunched') into the enzyme complex, presumably exactly as occurs during initial synthesis from the promoter; this synthesis then pauses at a defined site several nucleotides downstream from the active center position when σ70 first engages the -10 sequence repeat.

Two transcription pause elements underlie a σ70-dependent pause cycle.

The movement of RNA polymerase (RNAP) during transcription elongation is modulated by DNA-encoded elements that cause the elongation complex to pause. One of the best-characterized pause sequences is a binding site for the σ(70) initiation factor that induces pausing at a site near lambdoid phage late-gene promoters. An essential component of this σ(70)-dependent pause is the elemental pause site (EPS), a sequence that itself induces transcription pausing throughout the Escherichia coli genome and underlies other complex regulatory pause elements, such as the ops and his operon pauses.

Regulation of promoter-proximal transcription elongation: enhanced DNA scrunching drives λQ antiterminator-dependent escape from a σ70-dependent pause.

During initial transcription, RNA polymerase remains bound at the promoter and synthesizes RNA without movement along the DNA template, drawing downstream DNA into itself in a process called scrunching and thereby storing energy to sever the bonds that hold the enzyme at the promoter. We show that DNA scrunching also is the driving force behind the escape of RNA polymerase from a regulatory pause of the late gene operon of bacteriophage λ, and that this process is enhanced by the activity of the Q(λ) antiterminator. Furthermore, we show that failure of transcription complexes to escape the pause results in backtracking and arrest in a process analogous to abortive initiation.

High-resolution view of bacteriophage lambda gene expression by ribosome profiling.

Bacteriophage lambda is one of the most extensively studied organisms and has been a primary model for understanding basic modes of genetic regulation. Here, we examine the progress of lambda gene expression during phage development by ribosome profiling and, thereby, provide a very-high-resolution view of lambda gene expression. The known genes are expressed in a predictable fashion, authenticating the analysis.

A backtrack-inducing sequence is an essential component of Escherichia coli σ(70)-dependent promoter-proximal pausing.

RNA polymerase of both bacteria and eukaryotes can stall or pause within tens of base pairs of its initiation site at the promoter, a state that may reflect important regulatory events in early transcription. In the bacterial model system, the σ(70) initiation factor stabilizes such pauses by binding a downstream repeat of a promoter segment, especially the '-10' promoter element. We first show here that the '-35' promoter element also can stabilize promoter-proximal pausing, through interaction with σ(70) region 4.

Σ(70)-dependent transcription pausing in Escherichia coli.

After promoter escape in Escherichia coli, the initiating σ(70) factor is retained by core RNA polymerase (RNAP) for at least tens of nucleotides. While it is bound, σ(70) can engage a repeat of a promoter DNA element located downstream of the promoter and thereby induce a transcription pause. The σ(70)-dependent promoter-proximal pause that occurs at all lambdoid phage late gene promoters is essential to regulation of the late gene operons.

Promoter-specific control of E. coli RNA polymerase by ppGpp and a general transcription factor.

The stringent response of Escherichia coli reflects a global influence of the nucleotide ppGpp on gene expression in response to nutrient starvation. For critical elements of the response, the target of ppGpp is RNA polymerase, which can be either repressed or activated in a promoter-dependent way. A small protein, DksA, which binds in the secondary channel of RNA polymerase, possibly along with other general regulatory factors, contributes to ppGpp-dependent transcription regulation.

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.

A transcription antiterminator constructs a NusA-dependent shield to the emerging transcript.

The universal bacterial transcription elongation factor NusA mediates elongation activities of RNA polymerase. By itself, NusA induces transcription pausing and facilitates intrinsic termination, but NusA also is a cofactor of antiterminators that antagonize pausing and prevent termination. We show that NusA is required for lambda-related phage 82 antiterminator Q(82) to construct a stable complex in which RNA-based termination mechanisms have restricted access to the emerging transcript; this result suggests a locale for both Q(82) and NusA near the beta flap domain of RNA polymerase.

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.

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).

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.

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.

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.

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.

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.