Fidelity of RNA polymerase II transcription: Role of Rpb9 [corrected] in error detection and proofreading

Biochemistry. 2013 Nov 5;52(44):7807-17. doi: 10.1021/bi4009566. Epub 2013 Oct 24.

Abstract

The role of the small RNA polymerase II subunit Rpb9 in transcriptional proofreading was assessed in vitro. Transcription elongation complexes in which the 3' end of the RNA is not complementary to the DNA template have a dramatically reduced rate of elongation, which provides a fidelity checkpoint at which the error can be removed. The efficiency of such proofreading depends on competing rates of error propagation (extending the RNA chain without removing the error) and error excision, a process that is facilitated by TFIIS. In the absence of Rpb9, the rate of error propagation is increased by 2- to 3-fold in numerous sequence contexts, compromising the efficiency of proofreading. In addition, the rate and extent of TFIIS-mediated error excision is also significantly compromised in the absence of Rpb9. In at least some sequence contexts, Rpb9 appears to enhance TFIIS-mediated error excision by facilitating efficient formation of a conformation necessary for RNA cleavage. If a transcription error is propagated by addition of a nucleotide to the mismatched 3' end, then the rate of further elongation increases but remains much slower than that of a complex with a fully base-paired RNA, which provides a second potential fidelity checkpoint. The absence of Rpb9 also affects both error propagation and TFIIS-mediated error excision at this potential checkpoint in a manner that compromises transcriptional fidelity. In contrast, no effects of Rpb9 on NTP selectivity were observed.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Kinetics
  • Protein Subunits / chemistry
  • Protein Subunits / genetics
  • Protein Subunits / metabolism
  • RNA Polymerase II / chemistry
  • RNA Polymerase II / genetics
  • RNA Polymerase II / metabolism*
  • Saccharomyces cerevisiae / chemistry
  • Saccharomyces cerevisiae / enzymology*
  • Saccharomyces cerevisiae / genetics*
  • Saccharomyces cerevisiae Proteins / chemistry
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Transcription, Genetic*
  • Transcriptional Elongation Factors / chemistry
  • Transcriptional Elongation Factors / genetics
  • Transcriptional Elongation Factors / metabolism

Substances

  • Protein Subunits
  • Saccharomyces cerevisiae Proteins
  • Transcriptional Elongation Factors
  • transcription factor S-II
  • RNA Polymerase II