Intrinsic Transcript Cleavage in Yeast RNA Polymerase II Elongation Complexes

Abstract

Transcript elongation can be interrupted by a variety of obstacles, including certain DNA sequences, DNAbinding proteins, chromatin, and DNA lesions. Bypass of many of these impediments is facilitated by elongation factor TFIIS through a mechanism that involves cleavage of the nascent transcript by the RNA polymerase II/TFIIS elongation complex. Highly purified yeast RNA polymerase II is able to perform transcript hydrolysis in the absence of TFIIS. The "intrinsic" cleavage activity is greatly stimulated at mildly basic pH and requires divalent cations. Both arrested and stalled complexes can carry out the intrinsic cleavage reaction, although not all stalled complexes are equally efficient at this reaction. Arrested complexes in which the nascent transcript was cleaved in the absence of TFIIS were reactivated to readthrough blocks to elongation. Thus, cleavage of the nascent transcript is sufficient for reactivating some arrested complexes. Small RNA products released following transcript cleavage in stalled ternary complexes differ depending upon whether the cleavage has been induced by TFIIS or has occurred in mildly alkaline conditions. In contrast, both intrinsic and TFIIS-induced small RNA cleavage products are very similar when produced from an arrested ternary complex. Although ␣-amanitin interferes with the transcript cleavage stimulated by TFIIS, it has little effect on the intrinsic cleavage reaction. A mutant RNA polymerase previously shown to be refractory to TFIIS-induced transcript cleavage is essentially identical to the wild type polymerase in all tested aspects of intrinsic cleavage.Gene expression can be controlled by regulating any step of the transcription cycle: promoter binding, initiation, promoter escape, elongation, or termination. After transcript synthesis begins, the ability to suppress or complete the synthesis of an RNA transcript is vital to the cell, and a substantial and growing number of genes have been reported to be regulated during transcript elongation (1).The ternary elongation complex consists of the RNA polymerase, the template DNA, and the nascent RNA transcript. Surratt et al. (2) discovered that bacterial RNA polymerase ternary complexes have a mechanism for transcript shortening, endonucleolytic cleavage near the 3Ј-end of the transcript. After transcript cleavage, the 3Ј-fragment is released while the 5Ј-fragment is retained in an active ternary complex. A single cleavage event can liberate from 1 to 17 nt 1 of RNA bearing a 5Ј-monophosphate (3-6). Remarkably, transcription accurately resumes from the site of transcript cleavage to re-synthesize the excised RNA.Transcript cleavage activity is conserved in many DNA-dependent RNA polymerases, including bacterial RNA polymerases, vaccinia virus RNA polymerase (7), RNA polymerase I (8, 9), RNA polymerase II (10 -14), and RNA polymerase III (15). Accessory factors that stimulate transcript cleavage in ternary complexes have been identified in both prokaryotes (GreA and GreB) and eukaryotes (reviewed in Refs. 3,9,[16][17]...

Functional architecture of RNA polymerase I

“…Although the long linker between the A12.2 N-and C-terminal domains could in principle allow swinging of the C-terminal domain into the pore, our results suggest that the effect of A12.2 truncation on cleavage is due to an allosteric rearrangement in the Pol I active center. The conserved polymerase active site is capable of RNA cleavage in the absence of cleavage stimulatory factors, since free Pol II and the bacterial RNA polymerase can cleave RNA under mild alkaline conditions (Orlova et al, 1995;Weilbaecher et al, 2003). Consistently, the intrinsic cleavage activity of Pol I increased with increasing pH ( Figure 5C).…”

Control of Transcriptional Fidelity by Active Center Tuning as Derived from RNA Polymerase Endonuclease Reaction*

“…The absence of an effect of ␣-amanitin (which blocks trigger loop transition), on endonuclease cleavage in yeast pol II (41), as well as insensitivity of the same reaction to trigger loop deletion in E. coli (40) argues against the involvement of this transition in endonuclease reaction at least in the E. coli and yeast enzymes. In accordance with this conclusion, deletion of the entire trigger loop did not affect substrate discrimination by RNAP (40).…”

“…coli, and T. aquaticus RNAP, respectively (17,41, and this study). Contrary to E. coli, the endonuclease reaction in yeast pol II enzyme can yet proceed through the pyrophosphorolytic pathway (27).…”

A negative elongation factor for human RNA polymerase II inhibits the anti-arrest transcript-cleavage factor TFIIS

“…Although TFIIS stimulates transcript cleavage by stabilizing Mg 2ϩ II binding, RNAPII can catalyze the same cleavage reaction without TFIIS at slower rates (33). The rate of intrinsic cleavage can be increased by high pH, substitution of Mn 2ϩ for Mg 2ϩ , or high concentrations of Mg 2ϩ (26,33,34). We reasoned that substitution of Mn 2ϩ for Mg 2ϩ had the least potential to perturb RNAPII or DSIF͞ NELF.…”