Late sigma factor of bacteriophage T4. Formation and properties of RNA polymerase-promoter complexes.
Bacteriophage T4 late gene promoters do not display sequence homology in the -35 region (Christensen, A. C., and Young, E. T. (1982) Nature 299, 369-371), suggesting an unusual geometry of RNA polymerase-promoter interaction. We have analyzed in vitro utilization of a late T4 promoter by RNA polymerase reconstituted from E. coli core enzyme (E) and bacteriophage T4 late sigma factor (sigma gp55). The E sigma gp55 holoenzyme forms a stable promoter complex which lacks protein-DNA contacts upstream from position -30 and is sensitive to direct attack by heparin. This complex is capable of reiterative oligonucleotide synthesis (abortive initiation). Kinetic analysis of complex formation reveals a rapidly forming inactive intermediate (closed complex) which is slowly isomerized into a catalytically active form (open complex). The results indicate that all components essential for promoter binding, open complex formation, and initiation of transcription are present in the "downstream" part of the RNA polymerase molecule, which is defined by the -30 to +20 footprint. On the basis of these observations and the results of others, we suggest that during transcription initiation at bipartite (Escherichia coli) promoters, an "upstream" DNA-binding domain of RNA polymerase which recognizes specific sequence elements in the -35 region plays an auxiliary role by regulating the rate of productive interactions in the downstream part of the molecule through an allosteric mechanism.
Malik
S
S
Department of Microbiology, Columbia University College of Physicians and Surgeons, New York, New York 10032.
Goldfarb
A
A
D001483:Base Sequence; D004268:DNA-Binding Proteins; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D050981:Erythroid-Specific DNA-Binding Factors; D006493:Heparin / Q000494:pharmacology; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D013604:T-Phages / Q000235:genetics*; D014157:Transcription Factors
D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D014157:Transcription Factors; D006493:Heparin; D012321:DNA-Directed RNA Polymerases