Janice Pero

Nucleotide sequences that signal the initiation of transcription and translation inBacillus subtilis

SummaryWe have determined the nucleotide sequence of twoBacillus subtilis promoters (veg andtms) that are utilized by the principal form ofB. subtilis RNA polymerase found in vegetative cells (σ55-RNA polymerase) and have compared our sequences to those of several previously reportedBacillus promoters. Hexanucleotide sequences centered approximately 35 (the “-35” region) and 10 (the “-10” region) base pairs upstream from theveg andtms transcription startpoints (and separated by 17 base pairs) corresponded closely to the consensus hexanucleotides (TTGACA and TATAAT) attributed toEscherichia coli promoters. Conformity to the preferred -35 and -10 sequences may not be sufficient to promote efficient utilization byB. subtilis RNA polymerase, however, since three promoters (veg, tms andE. coli tac) that conform to these sequences and that are utilized efficiently byE. coli RNA polymerase were used with highly varied efficiencies byB. subtilis RNA polymerase.We have also analyzed mRNA sequences in DNA located downstream from eightB. subtilis chromosomal and phage promoters for nucleotide sequences that might signal the initiation of translation. In accordance with the rules of McLaughlin, Murray and Rabinowitz (1981), we observe mRNA nucleotide sequences with extensive complementarity to the 3′ terminal region ofB. subtilis 16S rRNA, followed by an initiation codon and an open reading frame.

Location of the phage λ gene responsible for turning off λ-exonuclease synthesis

Abstract A phage lambda gene, called tof , responsible for the turnoff of λ-exonuclease synthesis during the lytic cycle has been located in the region of the lambda genome which is nonhomologous with the λi 434 genome. The corresponding region of the λi 434 genome also contains a tof gene whose product turns off the synthesis of λ-exonuclease during the lytic cycle of phage λi 434 . The site of action of both tof gene products is also located in the region of nonhomology between the two phage genomes.

Nucleotide sequence of a promoter recognized by Bacillus subtilis RNA polymerase.

SummaryWe report the nucleotide sequence of a promoter recognized by RNA polymerase from the gram-positive bacterium Bacillus subtilis. This promoter, which was isolated from B. subtilis phage SP01 DNA, is homologous to promoters for Escherichia coli RNA polymerase; the sequences of the “-35 region” and the “Pribnow box” were 5′TTGACT and 5′CATAAT, respectively (T is the thymine analog 5-hydroxymethyluracil in SP01 DNA). These sequences each differed by only a single base pair from the preferred sequences for E. coli promoters. Not surprisingly, the SP01 promoter was actively transcribed in vitro by E. coli RNA polymerase as well as by B. subtilis RNA polymerase.

Conserved nucleotide sequences in temporally controlled bacteriophage promoters

Abstract Gene expression at a middle time in the lytic cycle of Bacillus subtilis bacteriophage SP01 is controlled by the product of phage gene 28 (gp28). gp28 is a sigma-like regulatory protein that directs the bacterial core RNA polymerase to bind and initiate transcription from promoter sites for phage middle genes. Here we report on the location, orientation and nucleotide sequences of five promoters in a cluster of middle genes on the phage genome. (The nucleotide sequences of two of these promoters were reported previously by Talkington & Pero, 1979). All five promoters shared highly conserved nucleotide sequences that were centered at about 35 and 10 base-pairs upstream from their start points of transcription. Based on these conserved sequences we propose that RNA polymerase containing gp28 recognizes the prototype sequence 5′T- -T-AGGAGA- -A-TT in the −35 promoter region and the sequence 5′TTT-TTT in the −10 region. (In SP01 DNA, T is the thymine analog 5-hydroxymethyluracil.) These prototype sequences differ strikingly from the corresponding conserved regions of SP01 early gene promoters which are recognized by the unmodified B. subtilis RNA polymerase and which are highly homologous to promoters for Excherichia coli RNA polymerase. These findings suggest that sigma factors (host sigma and gp28) dictate the recognition of both the −35 and −10 regions of promoters.

Bacteriophage SP01 regulatory proteins directing late gene transcription in vitro.

Expression of the temporally defined “late” genes of Bacillus subtilis phage SP01 requires the protein products of regulatory genes 33 and 34. These proteins have now been isolated in pure form. Together with a host factor delta, they act synergistically to direct “late” gene transcription in vitro by core RNA polymerase.

Regulatory gene 28 of bacteriophage SPO1 codes for a phage-induced subunit of RNA polymerase.

Abstract Phage SPO1 induces polypeptides that bind to Bacillus subtilis RNA polymerase and that apparently direct specific transcription in vitro. This report demonstrates that variants of one of these phage-induced polypeptides, termed IV (Mr 26,000), can be generated by suppression of a nonsense mutation in SPO1 regulatory gene 28. These variants differed in isoelectric point and were resolved by two-dimensional electrophoresis. This finding strongly suggests that gene 28 codes for a phage-induced subunit of RNA polymerase.

Restriction fragment analysis of the temporal program of bacteriophage SP01 transcription and its control by phage-modified RNA polymerases

Abstract The temporal program of phage SPO1 gene transcription and its regulation by modifications of RNA polymerase have been examined by using endonuclease restriction fragments of the phage genome as specific hybridization probes. Cells of Bacillus subtilis were pulsed-labeled at various times after infection by wild-type SPO1 or by mutants in regulatory gene 28, 33, or 34. Radioactive RNA from the pulse-labeled bacteria was then hybridized to electrophoretically separated restriction fragments of SP01-DNA. From the patterns of hybridization, we identified DNA fragments that contained early, middle, or late phage genes. To examine the control of phage gene transcription by modifications of RNA polymerase, RNA was copied in vitro from uncut SP01-DNA by either the unmodified B. subtilis transcriptase or by two forms of modified RNA polymerase that contained phage-coded regulatory subunits. RNA generated in vitro by each of the RNA polymerases exhibited a distinctive pattern of hybridization to the separated fragments of SPO1-DNA. RNA synthesized by unmodified host enzyme containing sigma factor preferentially annealed to DNA fragments containing early sequences, while RNA copied by the modified forms of RNA polymerase containing the gene 28 product or the products of genes 33 and 34 preferentially annealed to fragments containing middle and late genes, respectively. These findings confirm earlier reports indicating that the temporal program of SPOT gene expression is controlled by phage specified modifications in the transcriptional specificity of B. subtilis RNA polymerase.

Regulatory Subunits of RNA Polymerase

INTRODUCTION The discovery that the σ subunit of RNA polymerase governs site selection suggested a new model for the positive control of gene transcription. It was proposed by Burgess et al. (1969) that σ polypeptide could be replaced by regulatory proteins that would bind to the bacterial transcriptase and enable core enzyme to initiate transcription at new promoter sites. Reports of alterations in the transcriptional specificity of RNA polymerase in phage-infected (Seifert et al. 1969; Bautz and Dunn 1969; Travers 1969) and sporulating (Losick and Sonenshein 1969) bacteria generated considerable interest in this idea. Only recently, however, has it been possible to isolate purified RNA polymerases that contain known regulatory proteins and that direct specific gene transcription in vitro. Here we will review recent studies of regulatory subunits of RNA polymerase induced by phages T4, SPO1 and SP82, as well as several RNA polymerase binding proteins for which a transcriptional function has not yet been assigned. IDENTIFICATION OF RNA POLYMERASE BINDING PROTEINS The techniques of immunoprecipitation and affinity chromatography have made it possible to identify proteins that interact with RNA polymerase in crude extracts of bacteria and bacteriophage-infected cells. Proteins that bind to RNA polymerase can be precipitated by antibody directed against purified polymerase (see, for example, Greenleaf, Linn and Losick 1973) and will adhere to polymerase immobilized on agarose (Ratner 1974a). Ultimately, however, the identification of a new subunit of RNA polymerase requires the purification to homogeneity of the polymerase-subunit complex by conventional purification procedures and reconstitution of the...

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.

Bacteriophage SPO1 genes 33 and 34. Location and primary structure of genes encoding regulatory subunits of Bacillus subtilis RNA polymerase

Bacteriophage SPO1 gene 33 and 34 products are required for SPO1 late gene transcription. Both proteins bind to the core RNA polymerase of the Bacillus subtilis host to direct the recognition of SPO1 late gene promoters, whose sequences differ from those of SPO1 early and middle gene promoters. We have located and cloned the genes for these two regulatory proteins, and have engineered their expression in Escherichia coli by placing them under the control of the bacteriophage lambda PL promoter. Nucleotide sequence analysis indicated that genes 33 and 34 overlap by 4 base-pairs and encode highly charged, slightly basic proteins of molecular weight 11,902 and 23,677, respectively.