Transcription of cloned DNA from Bacillus subtilis phage SP01 requirement for hydroxymethyluracil-containing DNA by phage-modified RNA Polymerase

Abstract

We have cloned endonuclease restriction fragments of Bacillus subtilis phage SP01 DNA in a phage lambda vector, thereby replacing 5-hydroxymethyluracil (the thymine analog in SP01 DNA) with thymine. Two cloned fragments were shown to contain phage SP01 “early” genes. These cloned DNAs, as well as the 5-hydroxymethyluracil-containing fragments from which they were derived, supported specific RNA synthesis by B. subtilis RNA polymerase. Two other cloned DNAs contained phage “middle” genes, sequences that are known to be transcribed by a modified form of B. subtilis RNA polymerase containing a phage-coded regulatory protein (gp28) in place of the host sigma factor. The cloned middle genes failed to support specific RNA synthesis by phage-modified polymerase although the corresponding 5-hydroxymethyluracil-containing fragments were effective templates for in vitro transcription. This difference in the template activity of cloned and native SP01 fragments could not be attributed to promoter “mutations” introduced during replication in Escherichia coli as the nucleotide sequence of a middle gene promoter was identical for both thymine and 5-hydroxymethyluracil-containing DNA. We therefore conclude that, at least under certain conditions of in vitro RNA synthesis, 5-hydroxymethyluracil is required for specific transcription by gp28-containing RNA polymerase.