The T7 RNA polymerase intercalating hairpin is important for promoter opening during initiation but not for RNA displacement or transcription bubble stability during elongation.
The recently described crystal structures of a T7RNAP-promoter complex and an initial transcription complex reveal a beta-hairpin which inserts between the template and nontemplate strands of the promoter [Cheetham, G. M., et al. (1999) Nature 399, 80; Cheetham, G. M., et al. (1999) Science 286, 2305]. A stacking interaction between the exposed DNA bases and a valine at the tip of this hairpin may be especially important for stabilizing the opened promoter during initiation. It has been suggested that this hairpin may also be important for holding the transcription bubble open during transcript elongation, and a proposed model for how the RNA exits the transcription complex implies that this hairpin may also help displace the RNA from the template strand. To test these hypotheses, we have characterized both point and deletion mutants of this element. We find that these mutants exhibit reduced activity on linear, double-stranded templates but not on supercoiled or partially single-stranded templates. Probing of promoter-polymerase complexes, initial transcription complexes, and elongation complexes with KMnO(4) and a single-strand specific endonuclease reveals that the mutants have greatly reduced promoter unwinding activity during initiation. However, the structure and stability of the transcription bubble during elongation are not altered in the mutant enzymes, and RNA displacement activity is also normal. Thus, the T7RNAP intercalating hairpin is important, though not essential, for stabilizing the opened promoter during initiation, but is not important for RNA displacement or for transcription bubble structure or stability during elongation.
Brieba
L G
LG
Department of Biochemistry, University of Texas Health Sciences Center, San Antonio, Texas 78284-7760, USA.
Sousa
R
R
D017123:Bacteriophage T7 / Q000201:enzymology* / Q000235:genetics; D004278:DNA, Superhelical / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D043223:Deoxyribonuclease IV (Phage T4-Induced); D004706:Endodeoxyribonucleases / Q000737:chemistry; D004789:Enzyme Activation / Q000235:genetics; D004795:Enzyme Stability; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D010441:Peptide Chain Elongation, Translational / Q000235:genetics*; D010442:Peptide Chain Initiation, Translational / Q000235:genetics*; D011196:Potassium Permanganate / Q000737:chemistry; D011401:Promoter Regions, Genetic / Q000187:drug effects; D012330:RNA, Double-Stranded / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D013698:Templates, Genetic; D014158:Transcription, Genetic; D014764:Viral Proteins
D004278:DNA, Superhelical; D012330:RNA, Double-Stranded; D012367:RNA, Viral; D014764:Viral Proteins; D011196:Potassium Permanganate; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D004706:Endodeoxyribonucleases; D043223:Deoxyribonuclease IV (Phage T4-Induced)