Increased accommodation of nascent RNA in a product site on RNA polymerase II during arrest.

Abstract

RNA polymerases encounter specific DNA sites at which RNA chain elongation takes place in the absence of enzyme translocation in a process called discontinuous elongation. For RNA polymerase II, at least some of these sequences also provoke transcriptional arrest where renewed RNA polymerization requires elongation factor SII. Recent elongation models suggest the occupancy of a site within RNA polymerase that accommodates nascent RNA during discontinuous elongation. Here we have probed the extent of nascent RNA extruded from RNA polymerase II as it approaches, encounters, and departs an arrest site. Just upstream of an arrest site, 17-19 nucleotides of the RNA 3'-end are protected from exhaustive digestion by exogenous ribonuclease probes. As RNA is elongated to the arrest site, the enzyme does not translocate and the protected RNA becomes correspondingly larger, up to 27 nucleotides in length. After the enzyme passes the arrest site, the protected RNA is again the 18-nucleotide species typical of an elongation-competent complex. These findings identify an extended RNA product groove in arrested RNA polymerase II that is probably identical to that emptied during SII-activated RNA cleavage, a process required for the resumption of elongation. Unlike Escherichia coli RNA polymerase at a terminator, arrested RNA polymerase II does not release its RNA but can reestablish the normal elongation mode downstream of an arrest site. Discontinuous elongation probably represents a structural change that precedes, but may not be sufficient for, arrest by RNA polymerase II.Recent studies of the transcription elongation process have revealed that RNA chain synthesis is more complex than previously appreciated (1-8). At specific template sequences, RNA chain elongation by bacterial or eukaryotic RNA polymerases is uncoupled from translocation on DNA (1-8). The fraction of total transcription that takes place in this discontinuous or inchworm-like mode is unclear. The changing relationship between the RNA 3'-end and the elongation complex's DNA contacts has been mapped for a large collection of complexes at different positions on a single template (4). This survey suggested that inchworming alternates with periods of smooth chain assembly; during the latter process, base incorporation is coupled to translocation. It would appear that discontinuous elongation is provoked by specific DNA sequences and is accompanied by an unusual positioning of the RNA 3' terminus close to the complex's downstream boundary (3,4). This has been described as a "strained" (energetically unfavorable) configuration (4, 9, 10). Indeed, acquisition of the strained conformation appears to prepare the Escherichia coli elongation complex for termination (6, 7).Models of discontinuous elongation include an RNA product site in RNA polymerase that is alternately filled and emptied (5, 10, 11). An RNA-binding site has been observed on bacterial and eukaryotic RNA polymerases in which the RNA 3'-end is positioned near the enzyme's active site (12,13). In...

Nascent RNA structure modulates the transcriptional dynamics of RNA polymerases

“…The average maximum backtracked distance for Pol II in the presence of RNase A was 17 AE 4 bp. This number is consistent with bulk data that shows that 17-19 RNA base pairs are protected from digestion during transcription (32). According to this interpretation, the AT-rich nascent transcript does not organize sufficient secondary structure to prevent backtracking by the enzyme; eliminating most, but not all, of the secondary structure outside the polymerase makes the remaining transcript behave as an AT-rich nascent chain.…”

Interactions between DSIF (DRB sensitivity inducing factor), NELF (negative elongation factor), and the Drosophila RNA polymerase II transcription elongation complex

“…These results are consistent with the finding that human DSIF and NELF require transcripts ≥18 nt long to inhibit transcription (31), and also a recent study showed human DSIF preferentially bound elongation complexes containing transcripts that were at least 25 nt long (32). The 5′ end of an 18-nt-long nascent transcript just begins to emerge from the surface of Pol II (28,33). Exposure of four additional nucleotides appears to be sufficient for binding of DSIF alone or with NELF.…”

Mechanism of Poly(A) Signal Transduction to RNA Polymerase II In Vitro

“…Additional studies in vivo suggest that capping occurs when the RNA is between 25 and 30 nt long (69). Since approximately 18 nt of RNA is contained within the structure of the RNAPII ternary complex (39), capping must occur on 5Ј ends that lie only about 10 nt beyond the point of extrusion. Thus, functionally, the CTD appears to be designed to deliver capping enzyme and possibly other proteins to a region very close to the point at which the RNA emerges from the polymerase.…”