Distinguishing core and holoenzyme mechanisms of transcription termination by RNA polymerase III

Abstract

Transcription termination by RNA polymerase (Pol) III serves multiple purposes; it delimits interference with downstream genes, forms 3' oligo(U) binding sites for the posttranscriptional processing factor, La protein, and resets the polymerase complex for reinitiation. Although an interplay of several Pol III subunits is known to collectively control these activities, how they affect molecular function of the active center during termination is incompletely understood. We have approached this using immobilized Pol III-nucleic acid scaffolds to examine the two major components of termination, transcription pausing and RNA release. This allowed us to distinguish two mechanisms of termination by isolated Saccharomyces cerevisiae Pol III. A core mechanism can operate in the absence of C53/37 and C11 subunits but requires synthesis of 8 or more 3' U nucleotides, apparently reflecting inherent sensitivity to an oligo(rU·dA) hybrid that is the termination signal proper. The holoenzyme mechanism requires fewer U nucleotides but uses C53/37 and C11 to slow elongation and prevent terminator arrest. N-terminal truncation of C53 or point mutations that disable the cleavage activity of C11 impair their antiarrest activities. The data are consistent with a model in which C53, C37, and C11 activities are functionally integrated with the active center of Pol III during termination.

Fig 1

Two mechanisms of Pol III termination. Tailed template transcription was done with WT Pol III or Pol IIIΔ and templates with a 9T or 7T terminator. Reactions were in the presence of 0.5 mM (each) ATP, GTP, and UTP with 0.025 mM [α-³²P]CTP. T, terminated products; RT, read-through transcripts. Increasing amounts of purified recombinant C53/37 was included in lanes 5 to 7.

Fig 2

Pol IIIΔ exhibits two termination deficiencies, release and arrest. (A) Schematic of templates used for elongation complex (EC) assembly. Templates 1 and 2 differ only in the position of the terminator (A9 on the template strand) relative to the RNA primer (gray line; an asterisk indicates 5′ ³²P). Template 1 has a terminator 12 nt from the RNA 3′ end, while template 2 terminator abuts the RNA primer. Template 3 differs from 2 in that its terminator is 7T (A7 on the template); the same RNA was used for the three templates. (B) Transcription (lanes 1 to 4) and cleavage (lanes 5 to 8) activities of ECs with Pol III-WT and Pol IIIΔ on template 1. R, released; B, bound; T, terminated; RT, read-through; CP, cleavage products. Lanes 1 to 4 were enlarged; transcript bands reflecting the 3′ rU length are indicated on the right. Termination efficiency (TE) and release efficiency at the terminator (RET) are listed below the lanes. (C) Same as panel B but using template 2. Enlargements of lanes 1, 2, 7, and 8 are on the right. (D) Quantitative lane tracing profiles of the T (termination) region of lanes 1 and 3 of panel B. Lengths of 3′ rU residues are indicated under the corresponding peaks. (E) Same as panel B but using template 3. TE was determined by the formula [(terminator-released RNA + terminator-bound RNA)/(terminator-released RNA + terminator-bound RNA + read-through RNA bound + read-through RNA released)] × 100. RET is defined by the formula [terminator-released RNA/(terminator-released RNA + terminator-bound RNA)] × 100.

Fig 3

Slowing elongation is insufficient to correct release deficiency of Pol IIIΔ. (A) EC transcription reaction of WT and Pol IIIΔ at different NTP concentrations. EC was assembled on template 1, and transcription was done with the NTP concentrations indicated above the lanes (in μM). Transcription reactions were for 30 min. R and B indicate released and bound fractions, respectively. P, paused/arrested transcripts; T, terminator. (B) Superimposed quantitative lane tracing profiles of lanes 1, 3, and 5 from panel A. The arrows with numbers reflect the number of 3′ U nucleotides of the nascent RNA for each peak.

Fig 4

Cleavage-active C11 cannot prevent terminator arrest by Pol IIIΔ. (A) Schematic of template and RNA used for assembly of a 2-nt-backtracked complex. The 3′ end of the 12-nt RNA has a 2-nt rU·dT mismatch. (B) Transcription and cleavage by backtracked ECs incubated with either buffer alone (lanes 1 to 6) or with recombinant C11 (lanes 7 to 12) for 20 min prior to addition of Mg²⁺ and/or NTPs. Lanes 1, 2, 7, and 8 show products of transcription reactions; lanes 3, 4, 9, and 10 show cleavage reactions; and lanes 6, 7, 11, and 12 show starting material after incubation with and without C11 as indicated above the lanes. R and B represent released and bound, respectively. Cleavage products (CP) are indicated with black lines. (C) Transcription reactions in the presence of C11 at different NTP concentrations as indicated; the same amount of C11 as that used for panel B was used. (D) Cleavage assay for Pol III-WT and Pol IIIΔ ECs with various numbers of backtracked residues. ECs were assembled with RNAs having various numbers of mismatches, and after washes, complexes were treated with Mg²⁺ and/or C11 as indicated above each lane.

Fig 5

C53/37 and C11 constitute the holoenzyme mechanism of Pol III termination. (A) The released fractions only after transcription by Pol III-WT and Pol IIIΔ on template 1 in the presence of C53/37 or C53/37 and C11 as indicated above the lanes. (B) Quantitative lane tracing profiles of fractions shown in panel A as indicated by the reaction components at the right. (C) Transcription reactions done with template 2 in the presence of C53/37 and C53/37/C11 as indicated. (D) Quantitative lane tracing profiles of released fractions (lanes 1, 3, 5, and 7) shown in panel C. (E) Quantitative lane tracing profiles of bound fractions (lanes 2, 4, 6, and 8) shown in panel C. The number of 3′ U residues each peak corresponds to is indicated above and below the profiles.

Fig 6

C11 cleavage activity and the C53 N-terminal region are required for antiarrest activity but not released transcript length reduction. (A) Cleavage assay for wild-type C11 and D91A · E92A mutant C11. Two-nucleotide-backtracked complexes were assembled as described for Fig. 4B, followed by incubation with WT (W; lanes 5 and 6) or mutant C11 (M; lanes 7 and 8). Reactions were initiated for Pol IIIΔ samples by addition of MgCl₂ (lanes 6 and 8). (B) Transcription reactions on template 2 ECs for Pol III-WT or Pol IIIΔ with or without addition of WT or mutant C11 and C53/37; W indicates wild-type C11, and M indicates C11-D91A · E92A. C53-NtΔ/C37 represents the truncation mutant of C53 that lacks the first 280 amino acids. Proteins were incubated with elongation complexes for 20 min prior to addition of NTPs (0.5 mM). (C) Quantitative lane tracing profiles of the lanes corresponding to released samples (odd-numbered lanes). (D) Quantitative lane tracing profiles of the lanes corresponding to bound (arrested) transcripts (even-numbered lanes).