Fifty-four suppressible mutants of bacteriophage 429 have been isolated with a variety of mutagens and assigned to eight complementation groups. Viralspecific protein synthesis in UV light-irradiated, nonsuppressing Bacillus subtilis 60084 was analyzed with exponential acrylamide gels. Four additional 029 10"1 PFU of 029+ in TM buffer (0.01 M Tris-0.01 M MgSO4, pH 7.4). The mixture was incubated at room temperature (24 C) with gentle stirring. Samples (0.05 ml) were removed at 10-min intervals, and the reaction was stopped by diluting 1:100 in TMI buffer (TM containing 1 ml of a solution of 1.
Seventeen bacteriophage 029 proteins were detected in ultraviolet lightirradiated Bacillus subtilis by autoradiography of polyacrylamide slab gels. The appearance of 429 proteins occurred either before or concomitantly with viral DNA replication. Viral proteins detected early in the infectious cycle consisted of nine polypeptides ranging from 5,200 daltons to 54,000 daltons. Two of the early proteins were identified as, respectively, the major capsid protein and the protein comprising the filaments which extend from the head of the virus.
Ribonucleic acid (RNA) synthesis primed by bacteriophage T4 or X deoxyribonucleic acid (DNA) with Bacillus subtilis RNA polymerase is severely inhibited by high ionic strength. In contrast, RNA synthesis on B. subtilis bacteriophage 2C, SPOl, or 029 DNA is only moderately affected under similar conditions. The basis of this inhibition lies in the inability of the enzyme to initiate RNA chains with adenosine triphosphate or guanosine triphosphate (ATP, GTP). Binding to templates and the rate of catalysis in high salt after initiation do not seem to be affected. Incorporation of 'y-32P-ATP and GTP under a variety of conditions suggests that the specificity of B. subtilis RNA polymerase is different from that of the Escherichia coli enzyme and that it recognizes few promoters on T4 and A DNA. Although B. subtilis RNA polymerase initiates RNA chains primarily with ATP or GTP, initiations with pyrimidines can occur on DNA molecules in which hydroxymethyluracil replaces thymine. RNA synthesis on denatured DNA does not seem to be inhibited by high ionic strength, and on native T4 or X DNA the inhibition of initiation at constant ionic strength is inversely but not linearly proportional to the ionic radii of cations used to stabilize bihelical DNA to denaturation.In Escherichia coli the interaction of ribonucleic acid (RNA) polymerase with viral promoters has been shown to require the sigma factor which operates in specifying the initiation of RNA chains before the formation of the first nucleotide bond (3,4,6,9,10,31,33). Recent work has indicated that the number of phage promoters recognized by the E. coli enzyme can be determined (4). This number varies with the template used and represents only a fraction of the total number of promoters on viral chromosomes (3,11,22). However, in spite of these recent advances, the molecular mechanisms involved in the interaction of transcription initiation factors, core RNA polymerase, and nucleotide sequences specifying initiation start signals for RNA synthesis remain poorly understood.In Bacillus subtilis, RNA polymerase extracted from cells approaching sporulation has been shown to undergo a structural change in one of the beta chains (16)(17)(18). This is accompanied by a change in the specificity of initiation. This change in template specificity cannot be overcome by supplying the altered core enzyme with vegetative initiation factor or with the sigma factor of E. coli. Thus, the specificity of initiation may reside in part with the recognition of nucleotide sequences by the core enzyme, and transcription initiation factors may operate in catalyzing or specifying the interaction between these sequences and the core enzyme.Although the subunit composition of vegetative B. subtilis RNA polymerase is similar to that of the E. coli enzyme, differences have been noted (2, 18). The polypeptide functionally equivalent to the E. coli sigma factor has a molecular weight of 57,000 instead of 90,000 daltons, and the peptide presumably equivalent to the 40,000 E. coli alpha chain is 45,...
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