THE LENGTH AND SEQUENCE COMPOSITION OF VESICULAR STOMATITIS-VIRUS INTERGENIC REGIONS AFFECT MESSENGER-RNA LEVELS AND THE SITE OF TRANSCRIPTINITIATION

In this study, we used a dicistronic vesicular stomatitis virus (VSV) minigenome to investigate the effects of either single or multiple nuc leotide insertions placed immediately after the nontranscribed interge nic dinucleotide of the M gene on VSV transcription. Both Northern blo t and primer extension analysis showed that the polymerase responded t o the inserted nucleotides in a sequence-specific manner such that som e insertions had no effect on mRNA synthesis from the downstream G gen e, nor on the site of transcript initiation, whereas other insertions resulted in dramatic reductions in transcript accumulation. Some of th ese transcripts were initiated at the wild-type site, while others ini tiated within the inserted sequence. We also examined the transcriptio nal events that occurred when a natural, tl-nucleotide intergenic regi on located between the G and L genes from the New Jersey (NJ) serotype of VSV was inserted into the minigenome gene junction, In contrast to the normal 25 to 30% attenuation observed for downstream transcriptio n at gene junctions containing the typical dinucleotide (3'-GA-5') int ergenic region, the NJ variant showed greater than 75% attenuation at the gene junction, In addition, the polymerase initiated transcription at two major start sites, one of which was located within the interge nic sequence. Collectively, these data suggest that the polymerase ''s amples'' the intergenic sequences following polyadenylation and termin ation of the upstream transcript by scanning until an appropriate star t site is found. One implication of a scanning polymerase is that the polymerase presumably switches states from a processive elongation mod e to a stuttering mode for polyadenylation to one in which no transcri ption occurs, before it reinitiates at the downstream gene. Our data s upport the hypothesis that sequences surrounding the intergenic region modulate these events such that appropriate amounts of each mRNA are synthesized.