Cleavage of the nascent transcript induced by TFIIS is insufficient to promote read-through of intrinsic blocks to elongation by RNA polymerase II.

Abstract

RNA polymerases encounter a variety of types of blocks to elongation during transcription in eukaryotic cells. At least one protein, THIS, can promote read-through of many types of blocks to elongation by RNA polymerase II, and this protein stimulates cleavage of the nascent transcript in stalled elongation complexes as a prelude to read-through. The C-terminal halfofthe TFIIS protein is sufficient for stimulating the cleavage and read-through reactions in vitro. To Recently it has been suggested that TFIIS may operate in part by inducing cleavage of the 3' end of the nascent transcript in the ternary elongation complex (14,16,18). This cleavage reaction allows the polymerase to read through a template region that is otherwise only partially permissive for elongation (15). In a similar functional reaction, prokaryotic factors GreA and GreB stimulate cleavage in association with Escherichia coli RNA polymerase (19,20).At any block to elongation, only a fraction of the elongating polymerases stops transcription while the remainder reads through the block even in the absence of added factors (7,8,15). For those stalled polymerases, the transcript cleavage event stimulated by TFIIS precedes read-through (15). Following cleavage, read-through itself was thought to involve the passive resynthesis of RNA by RNA polymerase. Since only a fraction of polymerases would read through the block in any one cycle, repetitive cycles of cleavage and resynthesis would be necessary for all RNA polymerases eventually to read through the block (21). This hypothesis would predict that promoting the cleavage reaction would be the primary if not the only function of TFIIS in such read-through reactions. However, the results described here suggest that the read-through mechanism involving TFIIS cannot be explained so simply, as mutant TFIIS proteins, which are essentially inactive for promoting read-through, nonetheless stimulate efficient cleavage in stalled ternary elongation complexes.EXPERIMENTAL PROCEDURES Materials. Inhibit-ACE was obtained from 5'--3', Inc.Nucleotides were obtained from Pharmacia. [a-32P]CTP (>400 Ci/mmol; 1 Ci = 37 GBq) was obtained from Amersham. Bio-Gel 30 columns were obtained from Bio-Rad. His-Bind metal chelation resin was from Novagen. RNA polymerase II was purified from calf thymus as described (22).Strains and Plasmids. E. coli strain CJ236 (durt, ung-) was used to prepare uracil-containing template DNA for mutagenesis (23). A fragment containing the last 171 amino acids of the open reading frame of HeLa TFIIS (24), fused to 6 histidines at its N terminus, was prepared using the polymerase chain reaction (PCR) (25). The PCR product was digested with Nde I and BamHI and then ligated into the 5.4-kb Nde I-BamHI-digested pET22b(+) (Novagen). This plasmid is referred to as pET22b(+)/ATFIIS, and the protein produced from this plasmid is referred to as ATFIIS.Production ofMutants. Two synthetic oligonucleotides were constructed to mutate the charged amino acids E173, E174 (creating TFIIS5) or K186, K1...

Combinatorial Control of Human RNA Polymerase II (RNAP II) Pausing and Transcript Cleavage by Transcription Factor IIF, Hepatitis δ Antigen, and Stimulatory Factor II

“…Readthrough of arrest sites has been monitored as an SII function (51)(52)(53)(54)(55). Some SII mutants with moderate RNA cleavage activity do not appear to stimulate readthrough, indicating that stimulating transcript cleavage may not be the sole function of SII (51,52). To our knowledge, ours is the first report of combinatorial suppression of pausing by SII and TFIIF and the first report that SII (with TFIIF) may be an allosteric regulator of RNAP II pausing.…”

“…SII does not stimulate overall elongation rates, either in the presence or absence of TFIIF, at high (6, 7) or low (data not shown) NTP concentrations. Readthrough of arrest sites has been monitored as an SII function (51)(52)(53)(54)(55). Some SII mutants with moderate RNA cleavage activity do not appear to stimulate readthrough, indicating that stimulating transcript cleavage may not be the sole function of SII (51,52).…”

Intrinsic Transcript Cleavage in Yeast RNA Polymerase II Elongation Complexes

“…However, some TFIIS mutants (25,35,40) and at least one RNA polymerase II mutant (RPII⌬9 (25, 41)) are able to stimulate the formation of cleavage products but not reactivate elongation complexes arrested at the T1a site. TFIIS is clearly necessary to stimulate the transcript cleavage reaction in elongation complexes containing RNA polymerase II at physiological pH, but the additional TFIIS-dependent step required for readthrough still is not clear.…”

“…For both the wild type polymerase and RPII⌬9, intrinsic cleavage was sufficient to promote readthrough. However, some mutants in TFIIS that stimulate cleavage in arrested ternary complexes nonetheless do not promote readthrough (25,35,40). The effect of these mutants led to the hypothesis that TFIIS not only stimulates cleavage but also promotes an additional conformational change needed for efficient readthrough.…”

In Vitro Activity of the Baculovirus Late Expression Factor LEF-5

“…A similar decrease was obtained with single point mutations of the zinc ligands and the acidic residues in the QTRxxDE loop. The remaining 30% of activity is significant, however, because in SII the zinc ligands and acidic loop residues are absolutely essential for activity and substitution of any one of these residues is not tolerated (4,15). One explanation for these observations is that LEF-5 is multifunctional, with one activity that maps to the zinc ribbon domain and another that maps to the N-terminal part of the protein.…”