Identification and characterization of a transcription pause site in rotavirus

In rotavirus, transcription of the 11 double-stranded RNA genome segments o ccurs within the structurally intact subviral particle, and nascent transcr ipts are released through channels penetrating the two capsid layers at the icosahedral vertices. To gain insight into the early molecular events in t ranscription, we used high-resolution polyacrylamide gel electrophoresis to investigate the length distribution of transcription products at various t imes following initiation. We observed that, in the subviral particle under normal conditions, transcript initiation and capping are followed by a mom entary pause in elongation after the addition of 6 to 7 nucleotides. In the absence of the capping reaction cofactor S-adenosylmethionine, conditions under which the rate of nucleotide incorporation is reduced, we observe a s ignificant decrease in the ratio of paused to full length transcripts. We p ropose that this pause site may represent the point at which specific molec ular events take place to facilitate processive elongation. Furthermore, ou r results indicate that the presence of specific ligands on the viral surfa ce, such as VP7 in the mature virion, inhibits polymerase function. From th e perspective of the viral replication cycle, this inhibition may serve to ensure that transcription occurs with greatest efficiency only after the vi rus has entered the cytoplasm and assumed the form of a double layered part icle.