A subcomplex of RNA polymerase III subunits involved in transcription termination and reinitiation

Abstract

While initiation of transcription by RNA polymerase III (Pol III) has been thoroughly investigated, molecular mechanisms driving transcription termination remain poorly understood. Here we describe how the characterization of the in vitro transcriptional properties of a Pol III variant (Pol IIIdelta), lacking the C11, C37, and C53 subunits, revealed crucial information about the mechanisms of Pol III termination and reinitiation. The specific requirement for the C37-C53 complex in terminator recognition was determined. This complex was demonstrated to slow down elongation by the enzyme, adding to the evidence implicating the elongation rate as a critical determinant of correct terminator recognition. In addition, the presence of the C37-C53 complex required the simultaneous addition of C11 to Pol IIIdelta for the enzyme to reinitiate after the first round of transcription, thus uncovering a role for polymerase subunits in the facilitated recycling process. Interestingly, we demonstrated that the role of C11 in recycling was independent of its role in RNA cleavage. The data presented allowed us to propose a model of Pol III termination and its links to reinitiation.

Figure 1

Pol IIIΔ lacks the C11, C37, and C53 subunits. (A) Growth of the control wild-type strain (C37HA), and of the C37HAΔCt and the rpc37-1 mutant strains at 30 and 34°C. C37 subunit expressed in each strain is illustrated schematically on the right. Dark gray—HA tag, ^*—point mutation. (B) Pol III purified from the control wild-type strain expressing an HA-tagged version of C37 (lane 1), from the C37HAΔCt mutant strain (lane 2), and from the SpC11-C37HA mutant strain (lane 3) were analyzed by electrophoresis on a 13% SDS–polyacrylamide gel and silver-staining. Subunits are indicated on the left, ^*—major contaminants. (C) Western blot analysis of Pol III (lanes 1, 2, and 3 as in B) with a mixture of antibodies directed against the HA epitope and the Pol III subunits C34 or C53. Anti-C34 antibodies are used to control even Pol III loading. (D) Rescue of the thermosensitivity of the mutant rpc37-1 strain with plasmids overexpressing C11, C37, or C53. Corresponding empty vectors are shown as controls. Spots are formed from 10 μl of cells incubated for 2 days.

Figure 2

The terminator-recognition defect of Pol IIIΔ is corrected by addition of the r(C37–C53) heterodimer but not by the individual C11, C37, or C53 subunits. (A) Schematic representation of the SUP4 terminator with the T1 and T2 blocks. (B) The products of in vitro transcription reactions carried out using the SUP4 template and either the Pol IIIwt (lane 1), the Pol IIIΔ (lane 2), or the Pol IIIΔ preincubated for 10 min with an excess of recombinant C37, C53, or C11 subunits (lanes 3, 4, and 5, respectively) were separated by electrophoresis and autoradiographed. (C) Reactions as in (B) were performed with Pol IIIwt (lane 1), Pol IIIΔ (lane 2), or Pol IIIΔ preincubated with increasing amounts of the r(C37–C53) complex (lanes 3–5). Position of full-length transcripts terminated at T1 or T2 is indicated.

Figure 3

The r(C37–C53) heterodimer decreases the transcription elongation rate of Pol IIIΔ. (A) Time course analysis of elongation of radiolabeled 17-mer transcripts contained within the purified stalled ternary complexes of either Pol IIIwt or Pol IIIΔ to the full-length SUP4-o-tRNA^Tyr. As indicated, Pol IIIΔ ternary complexes were either incubated or not with the r(C37–C53) complex for 10 min. Full-length SUP4-o-tRNA^Tyr transcripts terminated at T1 or T2 are indicated, as well as those accumulated at the transitory pause sites (P1–P4). (B) Full-length transcripts were quantified and the values plotted as a function of time.

Figure 4

Pol IIIΔ+r(C37–C53) properly terminates transcription but does not reinitiate. (A) The number of transcriptionally active ternary complexes formed by the quantities of Pol IIIwt (lane 1) and of Pol IIIΔ (lane 2) used in subsequent experiments was assessed by the formation of stalled complexes assembled on the SUP4 gene in the absence of GTP. Note that Pol IIIΔ incorporates an extra nucleotide at the 3′ end of the nascent RNA forming an 18-mer RNA, as previously reported (Chédin et al, 1998). (B) Time course analysis of SUP4 gene transcription performed with Pol IIIwt or with Pol IIIΔ+r(C37–C53). Single-round transcription was performed with Pol IIIwt in the presence of heparin for 5 or 30 min. (C) The amounts of full-length SUP4-o-tRNA^Tyr transcripts in the time course analysis shown in (B) were quantified and the values plotted as a function of time. SR—single round. (D) Analysis of transcript release. Full-length transcripts contained in the supernatants of transcription reactions performed on an immobilized SUP4 template were separated by denaturing polyacrylamide gel electrophoresis, autoradiographed, and quantified. Transcription was carried out with either Pol IIIwt in the presence of heparin (single-round conditions) or Pol IIIΔ preincubated with r(C37–C53), and the reactions sampled at the times indicated. The values are given as a % of the final level of transcript released (which was similar in the two reactions examined) and plotted against time.

Figure 5

The C11 subunit is required for Pol III transcription reinitiation independently of its function in RNA cleavage activity. (A) Time course analysis of SUP4 gene transcription performed with Pol IIIwt, Pol IIIΔ, or Pol IIIΔ+rC11+r(C37–C53). Single-round transcription was performed with Pol IIIwt in the presence of heparin. (B) The amounts of full-length SUP4-o-tRNA^Tyr transcripts of the time course analysis shown in (A) were quantified and the values plotted as a function of time. (C) Ternary complexes of Pol IIIΔ containing an 18-mer transcript were purified and incubated with wild-type rC11 or with mutant rC11^E92H, as indicated. RNA cleavage reaction was performed for 5 and 60 s and the products separated by polyacrylamide gel electrophoresis in denaturing conditions and autoradiographed. ^*—cleavage products. (D) SUP4 gene was transcribed by Pol IIIΔ preincubated with r(C37–C53) alone, or with r(C37–C53) and either wild-type rC11 or the cleavage-incompetent rC11^E92H. Transcripts were separated by denaturing polyacrylamide gel electrophoresis and autoradiographed.

Figure 6

Pol IIIΔ reconstituted with rC11 and the r(C37–C53) heterodimer performs facilitated reinitiation. (A) Diagram describing the template competition experiment. (B) The GLU3 and SUP4 tRNA genes were separately preincubated with TFIIIC and TFIIIB to allow for the formation of stable preinitiation complexes. A limiting amount (50 ng) of wild-type Pol III or Pol IIIΔ+rC11+r(C37–C53) was then added for 15 min to one of the two complexes (template 1) (lanes 1, 2, 4, and 6), or to a mixture of the two preinitiation complexes (lanes 3 and 5). Incubation was continued for 5 min after addition of ATP (600 μM), CTP (600 μM), UTP (100 μM), and [α³²P]UTP (40 μCi), and then GTP (600 μM) was added alone (lanes 1, 2, 3, and 5), or together with the competitor, preassembled template 2 (lanes 4 and 6). Transcription was allowed to proceed for 5 min. The position of the two primary transcripts is indicated.

Figure 7

A model of Pol III transcription termination. (A) Transcription cycle of recycling wild-type Pol III performing facilitated reinitiation. During elongation, the preferential conversion of Pol III in a slow stepping state requires the presence of the C37–C53 heterodimer. This slow state allows the correct recognition of the terminator elements, and a C11-dependent conformational change of Pol III at the termination site required for reinitiation. (B) In the absence of the C37–C53 heterodimer, the enzyme is locked in a fast elongating-conformation and poorly recognizes terminator sequences. The enzyme is able to reinitiate transcription, but not to perform facilitated recycling. (C) In the absence of C11 subunit, the enzyme is blocked in the slow stepping state. At the terminator, the newly synthesized transcript is released, but the C11-dependent conformational change of Pol III cannot occur, and the enzyme stays in a noncompetent state for transcription reinitiation.