Transcriptional fidelity and proofreading in Archaea and implications for the mechanism of TFS-induced RNA cleavage.
We have addressed the question whether TFS, a protein that stimulates the intrinsic cleavage activity of the archaeal RNA polymerase, is able to improve the fidelity of transcription in Methanococcus. Using non-specific transcription experiments, we could demonstrate that misincorporation of non-templated nucleotides is reduced in the presence of TFS. A more detailed analysis revealed that elongation complexes containing a misincorporated nucleotide were arrested, but could be reactivated by TFS. RNase as well as exonuclease III footprinting experiments demonstrated that this arrest was not combined with extended backtracking. Analysis of paused elongation complexes demonstrated that TFS is able to induce a cleavage resynthesis cycle in such complexes, which resulted in the accumulation of dinucleotides corresponding to the last two nucleotides of the transcript. Further analysis of cleavage products revealed that, even under conditions that strongly promote misincorporation, still 50% of the released dinucleotides were correctly incorporated. Therefore, we assume that pausing of elongation complexes is an important determinant of TFS-induced RNA cleavage from the 3' end. As the incorporation rate of wrong nucleotides is about 700-fold reduced, it is possible that this delay also provides an appropriate time window for cleavage induction in order to maintain transcriptional fidelity by preventing misincorporation.
Lange
Udo
U
Institut für Allgemeine Mikrobiologie, Christian-Albrechts-Universität, Am Botanischen Garten 1-9, 24118 Kiel, Germany.
Hausner
Winfried
W
D001105:Archaea / Q000235:genetics / Q000378:metabolism*; D019843:Archaeal Proteins / Q000378:metabolism*; D001483:Base Sequence; D018983:DNA Footprinting; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000378:metabolism; D017017:Methanococcus / Q000235:genetics / Q000378:metabolism; D019642:RNA, Archaeal / Q000737:chemistry / Q000378:metabolism*; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism; D013698:Templates, Genetic; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic
D019843:Archaeal Proteins; D019642:RNA, Archaeal; D012333:RNA, Messenger; D014157:Transcription Factors; D012321:DNA-Directed RNA Polymerases; D005090:Exodeoxyribonucleases; D012260:Ribonucleases; C020133:exodeoxyribonuclease III