The RNA polymerase II elongation complex. Factor-dependent transcription elongation involves nascent RNA cleavage

Abstract

Regulation of transcription elongation is an important mechanism in controlling eukaryotic gene expression. SII is an RNA polymerase II-binding protein that stimulates transcription elongation and also activates nascent transcript cleavage by RNA polymerase II in elongation complexes in vitro (Reines, D. (1992) J. Biol. Chem. 267, 3795-3800). Here we show that SII-dependent in vitro transcription through an arrest site in a human gene is preceded by nascent transcript cleavage. RNA cleavage appeared to be an obligatory step in the SII activation process. Recombinant SII activated cleavage while a truncated derivative lacking polymerase binding activity did not. Cleavage was not restricted to an elongation complex arrested at this particular site, showing that nascent RNA hydrolysis is a general property of RNA polymerase II elongation complexes. These data support a model whereby SII stimulates elongation via a ribonuclease activity of the elongation complex.

FIG. 1. Activity of in vitro synthesized, recombinant mouse SII and truncated SII (ΔSII)

Panel A, RNA polymerase II binding assay. ³⁵S-Labeled, phosphocellulose-purified SII and ΔSII were resuspended at a concentration of 4 pM with 8WG16-immunoprecipitated rat liver RNA polymerase II at 28 °C for 15 min. Precipitable (P) and soluble ( S ) fractions were separated and run on a polyacrylamide gel. In control samples (lanes 5, 6, 9, and 10) anti-RNA polymerase II IgG was omitted. The amount of translation product put into each reaction (in) is displayed in lanes 1 and 2 for SII and ΔSII, respectively. Panel B, nascent transcript cleavage assay. Two μg of partially purified bovine brain SII (Br, lane 1 ) , in vitro synthesized mouse SII (lanes 2 and 3), in vitro synthesized mouse ΔSII (lanes 4 and 5), or buffer (−, lane 6 ) were mixed with washed elongation complexes and MgC1₂ and incubated for 30 min at 28 °C. The amount of in vitro synthesized SII or ΔSII added to each reaction is indicated by 1× , 2× , 5× , and 10× where × is approximately 50 amol. In the absence of nucleotides, the size of a cleaved nascent RNA is inversely proportional to the concentration of SII. Hence, shorter product was obtained in the presence of bovine brain (lane 1) compared to that seen with recombinant mouse SII (lanes 2 and 3) since more SII activity was present in the former than the latter. Runoff RNA (RO) and RNA resulting from transcription arrest at sire Ia (la) are indicated. Marker RNAs of 540, 420, 380 and nucleotides (top to bottom) are indicated with arrowheads.

FIG. 2. Time course of transcription through site Ia in the presence of SII and all four nucleotides

Six reaction equivalents of washed elongation complexes were incubated with bovine brain SII (12 μg) and 800 μM each of ATP, GTP, CTP, and UTP. After 20, 45,65, 95, and 900 s at 28 °C, one reaction equivalent was removed, and RNA was isolated for electrophoresis. Two shortened intermediates are indicated by an asterisk. The identity of the other RNAs are as indicated in the legend to Fig. 1B.

FIG. 3. Transcript shortening precedes SII-mediated transcription elongation

Panel A, RNA sequence around site Ia within the first intron of the human histone gene. The position of the 3′-ends formed when RNA polymerase II stops transcription are underlined and are collectively referred to as site Ia (Reines et al., 1987). The first A residue downstream of site Ia is boxed (□). The tentative site of cleavage which generates the major product (Reines, 1992) seen when washed elongation complexes were treated with SII (Fig. 3C, lane 1 ) is indicated by an arrow. Panel B, transcription through site Ia after the addition of SII, GTP, UTP, and CTP. Washed elongation complexes were incubated with bovine brain SII and 800μM each of UTP, CTP, and GTP. After 0, 0.5, 1,4,8, and 15 min at 28 °C, aliquots were withdrawn and labeled RNA was isolated for electrophoresis. The RNA that extends to the first A residue down-stream from site Ia is indicated (□). The migration position of other RNAs are as indicated in the legend to Fig. 1B. Panel C, transcription through site Ia in the presence of 3′-O-methyl-GTP. Washed elongation complexes were incubated with 800 μM each of UTP and CTP, bovine brain SII, and either 800 μM GTP (lane 2) or 830 μM 3′-O-methyl-GTP (lane 3), for 30 min at 28 °C. RNA was isolated and analyzed by electrophoresis and autoradiography. The migration position of transcript Ia, run in an adjacent lane, is shown (la). Other RNAs are identified as described in the legend to Fig. 3B.

FIG. 4. RNA polymerase II elongation complexes can carry out pyrophosphorolysis

Panel A, pyrophosphate-dependent transcript shortening. Washed elongation complexes were incubated for 30 min at 28 °C with 7 mM MgCl₂, and bovine brain SII (2 μg), or 1.8 mM sodium pyrophosphate as indicated above the figure. RNA was isolated and analyzed by electrophoresis and autoradiography. Panel B, effect of pyrophosphatase on transcript cleavage. Washed elongation complexes were mixed with 7 mM MgC1₂ and either bovine brain SII (2 μg, lanes 1 and 2) or 1.5 mM sodium pyrophosphate (lanes 3 and 4 ) in the presence (+) or absence (−) of 10 units of yeast inorganic pyrophosphatase. (One unit of inorganic pyrophosphatase liberates 1.0 μmol of inorganic orthophosphate/min.) The reactions were then incubated at 28 °C for 30 min before RNA was isolated for electrophoresis. Three pyrophosphatase-sensitive RNAs are indicated by asterisks.

FIG. 5. Other elongation complexes possess ,SII-activated nascent transcript cleavage activities

Panel A, an RNA polymerase II elongation complex arrested downstream of site Ia canc leave its nascent RNA. Elongation complexes arrested at site Ia were assembled and washed (lane 1 ) as described under “Materials and Methods.” Washed complexes were incubated at 28 °C with SII (lane 2) or SII and UTP, CTP, and GTP for 20 min (lanes 3–6) as described in the legend to Fig. 3B. These “ATP-starved” complexes (lanes 3–6) were washed by immunoprecipitation and incubated for 0 (lane 3), 0.5 (lane 4), 2 (lane5), or 10 min (lane 6) at 28 °C with approximately 5 × 10⁻⁴ absorbance units (280 nm) of rat liver SII (TSK-phenyl 5-PW fraction). Samples were prepared for electrophoresis and autoradiography as described under “Materials and Methods.” Panels B and C, runoff transcript cleavage. The plasmids pAdTerm-2 (B) or pDNAdML (C) were linearized with NdeI and transcribed in vitro with rat liver RNA polymerase II and general initiation factors. Reactions containing runoff RNAs (530 nt and 250 nt) pulse-labeled with [α-³²P]CMP were subjected to immunoprecipitation with anti-RNA monoclonal antibody. Precipitates were washed and incubated with approximately 5 × 10⁻⁴ absorbance units (280 nm) of rat liver SII (TSK-phenyl 5-PW fraction) and incubated at 28 °C. After the indicated number of minutes, aliquotsw ere withdrawn and RNAw as isolated for electrophoresis. An additional sample (chase) from each reaction was withdrawn (at 45 min, panel B, or at 60 min, panel C) and adjusted to 800 μM in all four nucleotides, incubated for an additional 15 miant 28 °C ,and RNwAa s prepared for electrophoresis. The migration position of marker RNAs of 540, 420, and 380 nucleotides (from top to bottom) are indicated by arrowheads in panel B. nt. nucleotides.