Abstract
Gene 4 of bacteriophage T7 encodes two proteins, a 63-kDa protein and a colinear 56-kDa protein, that are essential for synthesis of leading and lagging strands during DNA replication. The gene 4 proteins together catalyze the synthesis of oligoribonucleotides, pppACC(C/A) or pppACAC, at the single-stranded DNA sequences 3'-CTGG(G/T)-5' or 3'-CTGTG-5', respectively. Purified 56-kDa protein has helicase activity, but no primase activity. In order to study 63-kDa gene 4 protein free of 56-kDa gene 4 protein, mutations were introduced into the internal ribosome-binding site responsible for the translation of the 56-kDa protein. The 63-kDa gene 4 protein was purified 16,000-fold from Escherichia coli cells harboring an expression vector containing the mutated gene 4. Purified 63-kDa gene 4 protein has primase, helicase, and single-stranded DNA-dependent dTTPase activities. The constraints of primase recognition sequences, nucleotide substrate requirements, and the effects of additional proteins on oligoribonucleotide synthesis by the 63-kDa gene 4 protein have been examined using templates of defined sequence. A three-base sequence, 3'-CTG-5', is necessary and sufficient to support the synthesis of pppAC dimers. dTTP hydrolysis is essential for oligoribonucleotide synthesis. Addition of a 7-fold molar excess of 56-kDa gene 4 protein to 63-kDa protein increases the number of oligoribonucleotides synthesized by 63-kDa protein 100-fold. The increase in oligonucleotides results predominantly from an increase in the synthesis of tetramers, with relatively little change in the synthesis of dimers and trimers. The presence of 56-kDa protein also causes 63-kDa protein to synthesize "pseudo-templated" pppACCCC pentamers at the recognition sequence 3'-CTGGG-5'. T7 gene 2.5 protein, a single-stranded DNA binding protein, increases the total number of oligoribonucleotides synthesized by 63-kDa gene 4 protein on single-stranded M13 DNA, but has no effect on the ratio of dimers to trimers and tetramers.
Highlights
Background levels ofDNA synthesis activity by T7 DNA polymerase/thioredoxin complex in the absence of gene 4 proteins have been subtracted from the data.* dTTP hydrolysis activity in the absenceof single-stranded DNA hasbeen subtracted from the data
10%56-kDa proteinwas purified fromT 7 infected E. coli cells containing a mixture of 56- and63-kDa gene 4 proteins and Studier, 1983)
The 63-kDa gene 4 protein has been purified from E. coli
Summary
From the Departmentof Biolofical Chemistryand Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 0211.5. Pu- et al, 1983) and primase activities(Scherzinger et al, 1977a, rified 63-kDa gene 4 protein has primase, helicase, 1977b; RomanoandRichardson, 1979a, 1979b). The costs of publication of this article were activity and single-stranded DNA-dependentd T T P hydrolysisactivity,but is unabletocatalyzetemplate-dependent primer synthesis (Bernstein and Richardson, 1988a, 1988b). 10%56-kDa proteinwas purified fromT 7 infected E. coli cells containing a mixture of 56- and63-kDa gene 4 proteins and Studier, 1983). The 63-kDa protein cata- of the T7410 promoter (Fig. 1).The 5’-end of gene 4 was subcloned lyzed single-stranded DNA-dependent d T T P hydrolysis and from M13vector mGP4-lRBs to pGP4-6 aa1s.3-kb fragment resulting helicase activities (Bernstein and Richardson 989). The 63- from digestion with XbaI and SacI.Vector backbone and insert DNA kDa protein synthesized template-dependent primers, primase activity could be increased approximately 4-fold by the addition of a 7-fold excess of 56-kDa gene 4 protein. SDS-polyacrylamide gel electrophoresis of protein samples was carried out according to Laemmli (1970) in thepresence of 0.1% SDS
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