A negative elongation factor for human RNA polymerase II inhibits the anti-arrest transcript-cleavage factor TFIIS - PubMed (original) (raw)

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

Murali Palangat et al. Proc Natl Acad Sci U S A. 2005.

Abstract

Formation of productive transcription complexes after promoter escape by RNA polymerase II is a major event in eukaryotic gene regulation. Both negative and positive factors control this step. The principal negative elongation factor (NELF) contains four polypeptides and requires for activity the two-polypeptide 5,6-dichloro-1-beta-D-ribobenzimidazole-sensitivity inducing factor (DSIF). DSIF/NELF inhibits early transcript elongation until it is counteracted by the positive elongation factor P-TEFb. We report a previously undescribed activity of DSIF/NELF, namely inhibition of the transcript cleavage factor TFIIS. These two activities of DSIF/NELF appear to be mechanistically distinct. Inhibition of nucleotide addition requires > or = 18 nt of nascent RNA, whereas inhibition of TFIIS occurs at all transcript lengths. Because TFIIS promotes escape from promoter-proximal pauses by stimulating cleavage of back-tracked nascent RNA, TFIIS inhibition may help DSIF/NELF negatively regulate productive transcription.

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Figures

Fig. 1.

Fig. 1.

Comparison of effects of DSIF/NELF on TFIIS-stimulated transcript cleavage and on nucleotide addition. (A) Transcription templates. Sequences are shown for template 1 (wild-type HIV-1 transcript with the pause site indicated at U62) and template 2 (truncated HIV-1 transcript lacking anti-TAR and TAR with the pause site indicated at U28). Anti-TAR, dotted overline; TAR, black underline; truncation, open box. (B) Halted C27, U28, and U14 complexes (≈10 pM) were incubated with DSIF (≈60 nM) and NELF (≈7 nM) for 2 min at 30°C before addition of TFIIS (≈50 nM). Only a fraction of DSIF was active (14). Samples were removed at 8-s intervals and separated on a 12.5% denaturing gel (see Materials and Methods). The plots below each gel show the disappearance of C27, U28, and U14 RNA (•, +DSIF/NELF; ○, –DSIF/NELF). (C) TECs halted at U11–U14 on template 1 were elongated with all four NTPs (1 mM each). Aliquots were removed at 7-s intervals; a final sample (lane C) was removed 4 min after raising GTP to 5 mM. RNA products were analyzed as described (28), and the fraction U14 RNA with error analysis from three replicate experiments was plotted against reaction time. The entire gel is shown in Fig. 5, which is published as

supporting information

on the PNAS web site. (D) TECs halted at A26 formed on template 2 were elongated through the pause site in the presence of CTP, GTP, and UTP. Samples were collected, processed, and analyzed as described for C. (E) Ratio of pause strengths (product of pause duration and pause efficiency; refs. and 31) at different positions on template 1 in the presence and absence of DSIF/NELF represented as a bar graph. Pause strengths correspond to the areas under the curves in C and D and were calculated (Materials and Methods) for full time courses and RNA electrophoretograms, an example of which is shown in Fig. 5.

Fig. 2.

Fig. 2.

Inhibition of TFIIS requires both DSIF and NELF and appears to be competitive. (A) Halted U28 complexes were incubated with either DSIF (≈60 nM) or NELF (≈7 nM) or both for 2 min at 30°C before addition of TFIIS (≈50 nM) and removal of samples at 8-s intervals. Samples were separated on a 12.5% denaturing gel. (B) Competitive inhibition of TFIIS by DSIF/NELF. TFIIS-mediated transcript cleavage of C27 TECs was performed as in A with the indicated amounts of TFIIS in the presence of DSIF (≈9 nM) and NELF (≈3.5 nM). The rate of cleavage of C27 TECs (in s–1) is plotted as a function of TFIIS concentration (•, +DSIF/NELF; ○, –DSIF/NELF). (C) Increasing DSIF/NELF concentration does not increase TFIIS inhibition. Rates of transcript cleavage of C27 TECs were determined as described in the legend to B. TFIIS was at 500 nM. 1× DSIF was ≈9 nM; 1× NELF was ≈3.5 nM.

Fig. 3.

Fig. 3.

Intrinsic transcript cleavage activity of RNAPII is not inhibited by DSIF/NELF. U28 TECs were subjected to Mn2+-induced intrinsic transcript cleavage at 10 mM MnCl2, TFIIS-induced cleavage (50 nM as a control), or pyrophosphorolysis with 1 mM sodium pyrophosphate (PPi) in the absence or presence of DSIF/NELF. MnCl2 was replaced with 8 mM MgCl2 in the TFIIS and PPi reactions. The reaction conditions were otherwise as described in the legend to Fig. 2 A. Some lane-to-lane variation reflects loading differences caused by inhomogeneous sampling of the bead-immobilized TECs. The relative amounts of U28, C27, and A26 RNAs and the rates of U28 cleavage (0.04 ± 0.007 min–1) are indistinguishable with or without DSIF/NELF (Fig. 6).

Fig. 4.

Fig. 4.

Mechanism of DSIF/NELF inhibition of TFIIS. (A) Location of TFIIS binding to a TEC. A yeast RNAPII TEC model based on the crystal structure of Gnatt et al. (37) and the nucleic-acid-scaffold model of Korzheva et al. (36) is depicted with TFIIS located as reported by Kettenberger et al. (23). Portions of RNAPII (Left) are rendered semitransparent to reveal RNA, DNA, and TFIIS in the internal channels of the enzyme. (Left) The approximate length of exiting RNA; (Right) the distance between the exiting RNA and TFIIS. The location at which RPB1 CTD emerges from RNAPII (mobile magenta worm), the proposed SPT5-binding site (blue worm; ref. 39) on the RPB7 subunit (gray worm), the weakly bound Mg2+ ion required for transcript cleavage (yellow sphere; Mg2+II), and TFIIS (orange worm) are shown in both Left and Right. RPB4 is omitted for clarity. (B) Model depicting how DSIF/NELF inhibition of TFIIS could regulate promoter-proximal pausing. Double-headed arrow depicts possible NELF fluctuation (one of several ways to explain DSIF/NELF competition for TFIIS binding; see text). Red X depicts backtrack-paused RNAPII active site.

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