Mutations in the gene for a tRNA that functions as a regulator of a transcriptional attenuator in Bacillus subtilis.

It has been proposed that uncharged tRNA molecules may act as positive regulatory factors to control the expression of a number of operons in Bacillus subtilis and related bacteria by interacting with leader sequences to cause antitermination. In this study we report the isolation and characterization of regulatory mutations that modify one of the tRNA molecules predicted to have such a regulatory role. Three different alleles of the B. subtilis leucine tRNA gene leuG were found that resulted in higher expression of the ilv-leu biosynthetic operon. Each resulted in a base change in the D-loop of the leucine tRNA molecule with the anticodon 5'-GAG-3' (leucine tRNAGAG). Experiments with strains that are diploid for mutant and wild-type alleles suggested that both charged and uncharged tRNA molecules may interact with leader sequences to control expression of the operon.

The Bacillus subtilis ochre suppressor sup-3 is located in an operon of seven tRNA genes.

Most Bacillus subtilis tRNA genes have been isolated from lambda libraries by use of probes that hybridize to tRNA or rRNA sequences. None of those genes map to the region of the sup-3 mutation. By cloning of the sup-3 allele, a cluster of seven tRNA genes (the trnS operon) that had not been isolated by other methods was identified. In principle, this approach could be used to isolate at least one more predicted tRNA-containing operon in this bacterium. The trnS operon was shown to contain tRNA genes for Asn (GUU), Ser (GCU), Glu (UUC), Gln (UUG), Lys (UUU), Leu (UAG), and Leu (GAG). The sup-3 mutation was found to be a T-to-A transversion that changes the anticodon of the lysine tRNA from 5'-UUU-3' to 5'-UUA-3'. This result agrees with previous work that determined that the sup-3 mutation causes lysine to be inserted at ochre nonsense mutations.

Late promoter selection in the baculovirus gp64 envelope fusion protein gene.

The upstream promoter region of the Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) gp64 gene contains five copies of TAAG, the conserved sequence found at the transcriptional initiation sites of almost all baculovirus late genes. In AcMNPV-infected Sf9 cells, late transcription initiation is detected from only two upstream TAAG sites and not from three downstream TAAG sites. To examine several models for preferential TAAG site utilization, we constructed a series of recombinant AcMNPV baculoviruses that contain promoter region sequences from the gp64 gene fused to a chloramphenicol acetyl transferase reporter gene. Promoter-reporter constructs were inserted in the polyhedrin locus. To test a scanning model in which TAAG sites are sequentially selected according to their location in the region, we generated recombinant viruses in which the highly transcribed sites were inactivated by point mutations. Transcription from the mutant promoter constructs was compared qualitatively and quantitatively to transcription from the wild-type gp64 promoter. Inactivation of the upstream TAAG sites did not result in increased transcription from the downstream TAAG sites, suggesting that immediate context, rather than position, determines promoter utilization. To test this hypothesis, we made a series of minimal promoter constructs containing decreasing quantities of the sequences immediately flanking one of the active gp64 TAAG sites. Reporter constructs containing a gp64 TAAG site and > or = 12 bp of flanking sequence on both sides were transcribed at near wild-type levels. Constructs with less flanking sequence (9 or 6 bp of flanking sequence) were accurately transcribed, but at substantially lower levels, and transcription was not detected from constructs containing only 3 bp of flanking sequence. These results suggest that nucleotides immediately flanking the TAAG site (4-6 bp) are necessary for basal promoter activity while additional flanking sequences (> or = 12 bp) are required for late promoter activation and regulation. To further examine late promoter selection, we constructed recombinant AcMNPV baculoviruses that contain heterologous late promoters from the gp64 gene of the related virus Orgyia pseudotsugata MNPV (OpMNPV). TAAG sites that serve as functional late promoters in OpMNPV were found to mediate transcription initiation at only basal levels in the context of the AcMNPV genome, suggesting that late promoter activation may be virus specific within the family Baculoviridae.