Eric A. Galburt

Backtracking determines the force sensitivity of RNAP II in a factor-dependent manner

RNA polymerase II (RNAP II) is responsible for transcribing all messenger RNAs in eukaryotic cells during a highly regulated process that is conserved from yeast to human, and that serves as a central control point for cellular function. Here we investigate the transcription dynamics of single RNAP II molecules from Saccharomyces cerevisiae against force and in the presence and absence of TFIIS, a transcription elongation factor known to increase transcription through nucleosomal barriers. Using a single-molecule dual-trap optical-tweezers assay combined with a novel method to enrich for active complexes, we found that the response of RNAP II to a hindering force is entirely determined by enzyme backtracking. Surprisingly, RNAP II molecules ceased to transcribe and were unable to recover from backtracks at a force of 7.5 ± 2 pN, only one-third of the force determined for Escherichia coli RNAP. We show that backtrack pause durations follow a t-3/2 power law, implying that during backtracking RNAP II diffuses in discrete base-pair steps, and indicating that backtracks may account for most of RNAP II pauses. Significantly, addition of TFIIS rescued backtracked enzymes and allowed transcription to proceed up to a force of 16.9 ± 3.4 pN. Taken together, these results describe a regulatory mechanism of transcription elongation in eukaryotes by which transcription factors modify the mechanical performance of RNAP II, allowing it to operate against higher loads.

The origin of short transcriptional pauses.

RNA polymerases are protein molecular machines that transcribe genetic information from DNA into RNA. The elongation of the RNA molecule is frequently interrupted by pauses, the detailed nature of which remains controversial. Here we ask whether backtracking, the central mechanism behind long pauses, could also be responsible for short pauses normally attributed to the ubiquitous pause state. To this end, we model backtracking as a force-biased random walk, giving rise to a broad distribution of pause durations as observed in experiments. Importantly, we find that this single mechanism naturally generates two populations of pauses that are distinct both in duration and trajectory: long-time pauses with the expected behavior of diffusive backtracks, and a new class of short-time backtracks with characteristics similar to those of the ubiquitous pause. These characteristics include an apparent force insensitivity and immobility of the polymerase. Based on these results and a quantitative comparison to published pause trajectories measured with optical tweezers, we suggest that a significant fraction of short pauses are simply due to backtracking.

Double-stranded DNA translocase activity of transcription factor TFIIH and the mechanism of RNA polymerase II open complex formation

Significance How ATP hydrolysis is coupled to promoter DNA unwinding and open complex formation at RNA polymerase II (Poll II) promoters is a longstanding question. Of the multisubunit RNA polymerases, only Pol II requires ATP for DNA unwinding. Here we show that the general transcription factor TFIIH subunit Ssl2 is a double-stranded DNA translocase. These and other data suggest that Ssl2 promotes DNA opening by tracking along the nontemplate promoter strand, rotating and inserting DNA into the Pol II active site cleft, leading to DNA unwinding. Our accompanying biochemical studies explain why the open complex is unstable and how TFIIH can promote Pol II escape from the promoter. Our findings also have important implications for the mechanism of TFIIH-mediated DNA repair. Formation of the RNA polymerase II (Pol II) open complex (OC) requires DNA unwinding mediated by the transcription factor TFIIH helicase-related subunit XPB/Ssl2. Because XPB/Ssl2 binds DNA downstream from the location of DNA unwinding, it cannot function using a conventional helicase mechanism. Here we show that yeast TFIIH contains an Ssl2-dependent double-stranded DNA translocase activity. Ssl2 tracks along one DNA strand in the 5′ → 3′ direction, implying it uses the nontemplate promoter strand to reel downstream DNA into the Pol II cleft, creating torsional strain and leading to DNA unwinding. Analysis of the Ssl2 and DNA-dependent ATPase activity of TFIIH suggests that Ssl2 has a processivity of approximately one DNA turn, consistent with the length of DNA unwound during transcription initiation. Our results can explain why maintaining the OC requires continuous ATP hydrolysis and the function of TFIIH in promoter escape. Our results also suggest that XPB/Ssl2 uses this translocase mechanism during DNA repair rather than physically wedging open damaged DNA.

Single molecule transcription elongation

Single molecule optical trapping assays have now been applied to a great number of macromolecular systems including DNA, RNA, cargo motors, restriction enzymes, DNA helicases, chromosome remodelers, DNA polymerases and both viral and bacterial RNA polymerases. The advantages of the technique are the ability to observe dynamic, unsynchronized molecular processes, to determine the distributions of experimental quantities and to apply force to the system while monitoring the response over time. Here, we describe the application of these powerful techniques to study the dynamics of transcription elongation by RNA polymerase II from Saccharomyces cerevisiae.

CarD stabilizes mycobacterial open complexes via a two-tiered kinetic mechanism

CarD is an essential and global transcriptional regulator in mycobacteria. While its biological role is unclear, CarD functions by interacting directly with RNA polymerase (RNAP) holoenzyme promoter complexes. Here, using a fluorescent reporter of open complex, we quantitate RPo formation in real time and show that Mycobacterium tuberculosis CarD has a dramatic effect on the energetics of RNAP bound complexes on the M. tuberculosis rrnAP3 ribosomal RNA promoter. The data reveal that Mycobacterium bovis RNAP exhibits an unstable RPo that is stabilized by CarD and suggest that CarD uses a two-tiered, concentration-dependent mechanism by associating with open and closed complexes with different affinities. Specifically, the kinetics of open-complex formation can be explained by a model where, at saturating concentrations of CarD, the rate of bubble collapse is slowed and the rate of opening is accelerated. The kinetics and open-complex stabilities of CarD mutants further clarify the roles played by the key residues W85, K90 and R25 previously shown to affect CarD-dependent gene regulation in vivo. In contrast to M. bovis RNAP, Escherichia coli RNAP efficiently forms RPo on rrnAP3, suggesting an important difference between the polymerases themselves and highlighting how transcriptional machinery can vary across bacterial genera.

Mechanisms of backtrack recovery by RNA polymerases I and II

Significance Transcription of the genetic information from DNA into RNA is the central process of gene expression, and it is performed by enzymes called RNA polymerases (Pol). Transcription is interspersed with a proofreading mechanism called backtracking, during which the polymerase moves backward on the DNA template and displaces the RNA 3′ end from its active site. Backtrack recovery can happen by diffusion of the enzyme along the DNA or cleavage of the backtracked RNA. Using single-molecule optical tweezers and stochastic theory, we quantified distinct diffusion and cleavage rates of Pol I and Pol II and described distinct backtrack recovery strategies of these essential enzymes. During DNA transcription, RNA polymerases often adopt inactive backtracked states. Recovery from backtracks can occur by 1D diffusion or cleavage of backtracked RNA, but how polymerases make this choice is unknown. Here, we use single-molecule optical tweezers experiments and stochastic theory to show that the choice of a backtrack recovery mechanism is determined by a kinetic competition between 1D diffusion and RNA cleavage. Notably, RNA polymerase I (Pol I) and Pol II recover from shallow backtracks by 1D diffusion, use RNA cleavage to recover from intermediary depths, and are unable to recover from extensive backtracks. Furthermore, Pol I and Pol II use distinct mechanisms to avoid nonrecoverable backtracking. Pol I is protected by its subunit A12.2, which decreases the rate of 1D diffusion and enables transcript cleavage up to 20 nt. In contrast, Pol II is fully protected through association with the cleavage stimulatory factor TFIIS, which enables rapid recovery from any depth by RNA cleavage. Taken together, we identify distinct backtrack recovery strategies of Pol I and Pol II, shedding light on the evolution of cellular functions of these key enzymes.

CarD integrates three functional modules to promote efficient transcription, antibiotic tolerance, and pathogenesis in mycobacteria

Although the basic mechanisms of prokaryotic transcription are conserved, it has become evident that some bacteria require additional factors to allow for efficient gene transcription. CarD is an RNA polymerase (RNAP)‐binding protein conserved in numerous bacterial species and essential in mycobacteria. Despite the importance of CarD, its function at transcription complexes remains unclear. We have generated a panel of mutations that individually target three independent functional modules of CarD: the RNAP interaction domain, the DNA‐binding domain, and a conserved tryptophan residue. We have dissected the roles of each functional module in CarD activity and built a model where each module contributes to stabilizing RNAP–promoter complexes. Our work highlights the requirement of all three modules of CarD in the obligate pathogen Mycobacterium tuberculosis, but not in Mycobacterium smegmatis. We also report divergent use of the CarD functional modules in resisting oxidative stress and pigmentation. These studies provide new information regarding the functional domains involved in transcriptional regulation by CarD while also improving understanding of the physiology of M. tuberculosis.

His-Cys Box Homing Endonucleases

Homing endonucleases are often grouped into four families based on distinct sequence motifs. One of these families is known as the His-Cys box homing endonucleases and contains two clusters of conserved histidine and cysteine residues over a central 100 amino acid region. At last count, 23 members of this family had been identified. The open reading frames (ORFs) of these proteins are contained within mobile group I introns found in nuclear rDNA genes of several protists. The nuclear location of these introns and ORFs is currently unique among the homing endonuclease families and poses an intriguing puzzle regarding their expression from non-coding rRNA transcripts.

Force-dependent melting of supercoiled DNA at thermophilic temperatures

Local DNA opening plays an important role in DNA metabolism as the double-helix must be melted before the information contained within may be accessed. Cells finely tune the torsional state of their genomes to strike a balance between stability and accessibility. For example, while mesophilic life forms maintain negatively superhelical genomes, thermophilic life forms use unique mechanisms to maintain relaxed or even positively supercoiled genomes. Here, we use a single-molecule magnetic tweezers approach to quantify the force-dependent equilibrium between DNA melting and supercoiling at high temperatures populated by Thermophiles. We show that negatively supercoiled DNA denatures at 0.5 pN lower tension at thermophilic vs. mesophilic temperatures. This work demonstrates the ability to monitor DNA supercoiling at high temperature and opens the possibility to perform magnetic tweezers assays on thermophilic systems. The data allow for an estimation of the relative energies of base-pairing and DNA bending as a function of temperature and support speculation as to different general mechanisms of DNA opening in different environments. Lastly, our results imply that average in vivo DNA tensions range between 0.3 and 1.1 pN.

Time-resolved macromolecular crystallography

Rapid x-ray characterization of structure and innovative ways of initiating and controlling reactions are shedding new light on protein function by enabling the visualization of macromolecules in action.