Initiation of Bacillus subtilis Ribonucleic Acid Polymerase on Deoxyribonucleic Acid from Bacteriophages 2C, φ29, T4, and Lambda

Abstract

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,...

Transcription of the Genome of Bacteriophage φ29: Isolation and Mapping of the Major Early mRNA Synthesized In Vivo and In Vitro

“…It has been previously shown that the initiation of 429 RNA synthesis in vitro by B. subtilis RNA polymerase takes place mainly with GTP and to a lesser extent with ATP (26,38). Table 3 also shows that in vitro RNA initiated with [y-32P]GTP (pppG-RNA) hybridized exclusively with the EcoRI-A and -C fragments but not with the EcoRI-B fragment.…”