Transcription Processivity: Protein-DNA Interactions Holding Together the Elongation Complex

138

120

Escherichia coli RNA polymerase translocates along the DNA discontinuously during the elongation phase of transcription, spending proportionally more time at some template positions, known as pause and arrest sites, than at others. Current models of elongation suggest that the enzyme backtracks at these locations, but the dynamics are unresolved. Here, we study the role of lateral displacement in pausing and arrest by applying force to individually transcribing molecules. We find that an assisting mechanical force does not alter the translocation rate of the enzyme, but does reduce the efficiency of both pausing and arrest. Moreover, arrested molecules cannot be rescued by force, suggesting that arrest occurs by a bipartite mechanism: the enzyme backtracks along the DNA followed by a conformational change of the ternary complex (RNA polymerase, DNA and transcript), which cannot be reversed mechanically.E scherichia coli RNA polymerase (RNAP) is a highly processive enzyme responsible for the transcription of DNA into RNA. The ternary elongation complex of DNA, RNAP and RNA is extremely stable, with RNAP capable of reaching speeds of 35 nucleotides per second as it translocates along the DNA (1). Despite this rapid translocation during elongation, RNAP is sensitive to the sequence it transcribes, displaying temporary (pauses) and permanent (arrests) halts to transcription, which are believed to play a role in the regulation of gene expression (2).Transcriptional pauses and arrests can occur by various mechanisms but share common features. These features include the continued stability of the ternary elongation complex (3) and the displacement of the RNA 3Ј end from the enzyme active site (4, 5). Kinetic evidence suggests that both pauses and arrests are kinetically off the main elongation pathway, and that pauses are intermediate between elongation and arrest states (4, 6-8):Much of the information about pauses and arrests has come from biochemical footprinting and crosslinking studies on complexes walked to particular template positions. These studies have suggested that core RNA polymerase is, variously, a flexible enzyme, capable of undergoing inchworming motion (9-11); a rigid enzyme, capable of monotonic translocation (12); and a ''sliding clamp'' enzyme, capable of frequent backwards and forwards oscillations (13-15). Among these proposed models, the importance given to an RNA:DNA hybrid and to different protein-nucleic acid interactions to explain the stability of the complex varies (16). Within the sliding clamp model, proteinnucleic acid contacts confer stability to the ternary complex (through a protein clamp enclosing downstream DNA), whereas the RNA:DNA hybrid (of 8-12 bp) is responsible for accurately positioning the enzyme active site with the 3Ј OH of nascent RNA (13,15,17). The recently obtained crystal structure of a bacterial RNA polymerase shows evidence of a downstream DNAbinding site, and suggests that an RNA:DNA hybrid of 8-9 bp is easily accommodated within the enzyme, which is consistent wi...

Section: Effect Of Force On Pausingmentioning

confidence: 41%

Using mechanical force to probe the mechanism of pausing and arrest during continuous elongation by Escherichia coli RNA polymerase

Forde

Izhaky

Woodcock

et al. 2002

138

120

“…4 provides a framework for interpretation of genetic, biochemical, and structural data on RNAPII-dependent transcription initiation and regulation. Based on similarities between RNAPII and other multisubunit RNA polymerases (i.e., RNA polymerases I and III, E. coli RNA polymerase) in threedimensional structure (39,40), subunit primary structure (41,42), DNA crosslinking (43)(44)(45)(46)(47), DNA bending (48)(49)(50)(51), and DNA wrapping (51-54), we suggest that the model in Fig. 4 may apply generally to multisubunit RNA polymerases.…”

Section: Discussionmentioning

confidence: 99%

Trajectory of DNA in the RNA polymerase II transcription preinitiation complex

Kim

Lagrange

Wang

et al. 1997

111

124

By using site-specific protein-DNA photocrosslinking, we define the positions of TATA-binding protein, transcription factor IIB, transcription factor IIF, and subunits of RNA polymerase II (RNAPII) relative to promoter DNA within the human transcription preinitiation complex. The results indicate that the interface between the largest and second-largest subunits of RNAPII forms an extended, Ϸ240 Å channel that interacts with promoter DNA both upstream and downstream of the transcription start. By using electron microscopy, we show that RNAPII compacts promoter DNA by the equivalent of Ϸ50 bp. Together with the published structure of RNAPII, the results indicate that RNAPII wraps DNA around its surface and suggest a specific model for the trajectory of the wrapped DNA.Transcription initiation at a eukaryotic protein-encoding gene involves assembly on promoter DNA of a complex consisting of RNA polymerase II (RNAPII) and six general transcription factors: IIA, IIB, IID (or TATA-element binding protein, TBP), IIE, IIF, and IIH (1-3). A subcomplex containing TBP, IIB, IIF, RNAPII, and promoter DNA is stable (4, 5), and, under certain conditions (e.g., conditions that promote DNA melting), is fully competent for transcription initiation (6-11). Results of DNA footprinting experiments indicate that the TBP-IIB-IIF-RNAPII-promoter complex with linear DNA at 30°C involves interactions both upstream and downstream of the transcription start (positions Ϫ42 to ϩ17; H. Lu and D.R., unpublished data) and involves unmelted DNA (11). Thus, the TBP-IIB-IIF-RNAPII-promoter complex with linear DNA at 30°C appears to correspond to the RNA polymerase-promoter ''intermediate complex'' characterized in studies of Escherichia coli RNA polymerase (RP i or RP c2 ; refs. 12-15).The TBP-IIB-IIF-RNAPII-promoter complex contains at least 14 distinct polypeptides (one in TBP, one in IIB, two in IIF, and at least 10 in RNAPII) and has a molecular mass in excess of 700 kDa (1-3). High-resolution structures have been determined for the TBP-DNA and TBP-IIB-DNA complexes (16-18). However, the TBP-IIB-IIF-RNAPII-promoter complex is too large for high-resolution structure determination by current methods. Therefore, information about the structure of the TBP-IIB-IIF-RNAPII-promoter complex must rely on low-resolution structure determination (19,20) supplemented by biochemical and imaging data.In the work in this report, we have used site-specific protein-DNA photocrosslinking and electron microscopy to define protein-DNA interactions within the human TBP-IIB-IIF-RNAPIIpromoter complex. MATERIALS AND METHODSDerivatized Promoter DNA Fragments. Derivatized promoter DNA fragments were prepared essentially as in ref. 21. Oligodeoxyribonucleotides containing phosphorothioate 5Ј to the third nucleotide were synthesized by using solid-phase ␤-cyanoethylphosphoramidite chemistry and tetraethylthiuram disulfide (Applied Biosystems), purified on OPC (Applied Biosystems), and derivatized with azidophenacyl bromide (Sigma; ref. 22). Derivatized oligodeo...

“…Nudler et al suggest that stabilization of the initiating transcription complex upon growth of the nascent RNA to 8-10 nt reflects strengthening of the DNA clamp when RNA enters the exit channel, and that termination could involve loss of this stabilizing influence (41). Our finding of a shift in RNA interaction away from ␤Ј and to ␤904-950 is consistent with this idea and suggests that interaction of RNA with the N-terminal region of ␤Ј (which occurs at least for bases 12-17 nt upstream from the RNA 3Ј end in the absence of a hairpin; D.W., unpublished data) may stabilize the transcription elongation complex (Fig.…”

mentioning

confidence: 99%

“…Loss of RNA contact to this N-terminal segment of ␤Ј is interesting for several reasons. Together with the ␤ C-terminal region, ␤Ј1-81 forms a ''clamp'' that contacts DNA in front of the active site and confers stability to the elongating transcription complex (41,42). ␤Ј1-81 contains a C 4 Zn-finger-like motif (38), and both genetic studies (43) and the viability of a ␤::␤Ј fusion (K.S., S. A. Darst, R. A. Mooney, and R.L., unpublished work) suggest it lies near the C-terminal region of ␤ (which also contains a C 4 Zn-finger-like motif in eukaryotes but not in bacteria).…”

mentioning

confidence: 99%

Preferential interaction of the his pause RNA hairpin with RNA polymerase β subunit residues 904–950 correlates with strong transcriptional pausing

Wang¹,

Severinov²,

Landick³

1997

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 -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 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 ␤904-950 replaced crosslinking to ␤ and to other parts of ␤ that occurred in nonpaused complexes prior to hairpin formation. These changes in nascent RNA interactions may inhibit reactive alignment of the RNA 3 end in the paused complex and be related to events at -independent terminators.The his leader pause hairpin is a well characterized nascent RNA structure that modulates RNA chain elongation (1-4). It forms concomitantly with a paused conformation of the transcription complex midway through the his biosynthetic operon leader region, where it increases the lifetime of the paused complex at least 6-fold. The pause allows time for a ribosome to initiate synthesis of the leader peptide and release the paused polymerase (probably by disrupting the pause hairpin), thereby synchronizing ribosome and polymerase movement during transcriptional attenuation (5). This and other pause sites are regulatory timing mechanisms that both prevent transcription beyond a region where regulatory input is effective and put polymerase in the proper conformation to interact with regulatory molecules.Similar hairpins are involved in some, but not all, pause signals; are required at -independent terminators (6-8), where they trigger dissociation of the transcription complex; and alone (e.g., HK022 put; ref. 9) or in association with proteins (e.g., N-nut complex; ref. 8) can function as antiterminators, which modify the transcription complex to block recognition of both pause sites and terminators. Nascent RNA hairpins also are components of some eukaryotic regulatory mechanisms (e.g., HIV TAR RNA; ref. 10) and can trigger pausing or termination by RNA polymerase II in vitro (11). Although RNA hairpin-RNA polymerase interactions have long been suggested to play roles in pausing (12), termination (13-15), and antitermination (16), no direct evidence for these interactions or their effects exists.The his pause RNA hairpin offers an excellent paradigm to study these interactions. It is one of four components of the multipartite his pause signal that also depends on a 3Ј-proximal region of RNA or DNA, alignment of the 3Ј-terminal nucleotide and the incoming NTP, and duplex DNA that contacts polymerase downstream from the active site (1, 2). Both the his pause signal and -independent terminators ...