The role of the largest RNA polymerase subunit lid element in preventing the formation of extended RNA-DNA hybrid.
Analysis of multi-subunit RNA polymerase (RNAP) structures revealed several distinct elements that may perform partial functions of the enzyme. One such element, the "lid", is formed by an evolutionarily conserved segment of the RNAP largest subunit (beta' in bacterial RNAP). The beta' lid contacts the nascent RNA at the upstream edge of the RNA-DNA hybrid, where the RNA gets separated from the DNA template-strand and double-stranded upstream DNA is formed. To test the beta' lid functions, we generated bacterial RNAP lacking the lid and studied the mutant enzyme's properties in vitro. Our results demonstrate that removal of the lid has minimal consequences on transcription elongation from double-stranded DNA. On single-stranded DNA, the mutant RNAP generates full-sized transcripts that remain annealed to the DNA throughout their length. In contrast, the wild-type enzyme produces short, 18-22 nucleotide transcripts that remain part of the transcription complex but cannot be further elongated. The cessation of transcription is apparently triggered by a clash between the lid and the nascent RNA 5' end. The results show that the lid's function is redundant in the presence of the non-template DNA strand, which alone can control the proper geometry of nucleic acids at the upstream edge of the transcription complex. Structural considerations suggest that in the absence of the non-template strand and the lid, a new channel opens within the RNAP molecule that allows continuous DNA-RNA hybrid to exit RNAP.
Naryshkina
Tatyana
T
Waksman Institute, Rutgers, The State University, Piscataway, NJ 08854, USA.
Kuznedelov
Konstantin
K
Severinov
Konstantin
K
D000595:Amino Acid Sequence; D017123:Bacteriophage T7; D017124:Conserved Sequence; D004269:DNA, Bacterial / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009693:Nucleic Acid Hybridization; D011196:Potassium Permanganate / Q000378:metabolism; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D012329:RNA, Bacterial / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013698:Templates, Genetic; D013824:Thermus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D012329:RNA, Bacterial; D011994:Recombinant Proteins; D011196:Potassium Permanganate; C108570:beta' subunit of RNA polymerase; D012321:DNA-Directed RNA Polymerases