Processivity of proteolytically modified forms of T7 RNA polymerase.
Two proteolytically modified forms of T7 RNA polymerase have been characterized with respect to transcription initiation and processivity. One species, denoted 80K-20K, is singly cleaved within the region of the polypeptide chain between amino acids 172 and 180. The second species, denoted 80K, is generated by extensive proteolysis of the N-terminal 20K domain by trypsin. The 80K-20K form is fully active in initiation and escape from abortive cycling. It is deficient only in processivity on long DNA templates. Likewise, the 80K species shows initiation kinetics and abortive product synthesis similar to those of the native enzyme. This latter species, however, is unable to escape abortive cycling and shows no synthesis of transcripts longer than about eight bases. Studies of RNA and DNA binding to the three different forms of the enzyme by gel retention assays reveal that the native (98K), the 80K-20K, and the 80K species all form specific complexes with promoter-containing DNA. In addition, the native enzyme binds nonspecifically to double-stranded DNA, while the 80K-20K and 80K enzymes do not. The native enzyme also binds RNA. This RNA binding is reduced in the 80K-20K enzyme and is absent in the 80K species. We suggest a model for T7 RNA polymerase wherein the 20K N-terminal domain of the protein or a shared region between the N- and the C-terminal domains of the protein forms a nonspecific polynucleotide binding site.(ABSTRACT TRUNCATED AT 250 WORDS)
Muller
D K
DK
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06510.
Martin
C T
CT
Coleman
J E
JE
D004268:DNA-Binding Proteins / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012313:RNA / Q000378:metabolism; D013329:Structure-Activity Relationship; D013604:T-Phages / Q000201:enzymology*; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D012313:RNA; D012321:DNA-Directed RNA Polymerases; D010447:Peptide Hydrolases