Vaccinia virus early gene transcription termination factors VTF and Rap94 interact with the U9 termination motif in the nascent RNA in a transcription ternary complex

Abstract

The vaccinia virus core contains a 195 kb double stranded DNA genome, a multi-subunit RNA polymerase, transcription initiation and termination factors and mRNA processing enzymes. Upon infection, vaccinia virus early gene transcription takes place in the virus core. Transcription initiates at early promoters and terminates in response to a termination motif, UUUUUNU, in the nascent mRNA. Early gene transcription termination requires the vaccinia virus termination factor, VTF, a single stranded DNA-dependent ATPase, and NPH I, the Rap94 subunit of the virion RNA polymerase, as well as the presence of the UUUUUNU motif in the nascent RNA. The position of UUUUUNU in the ternary complex suggests that it serves as a site of interaction with one or more components of the transcription termination complex. In order to identify the factor(s) that interact with UUUUUNU a series of direct UV photo crosslinking and ribonuclease A protection studies were undertaken. Through these analyses both VTF and Rap94 were shown to interact with UUUUUNU in the isolated ternary complex. Evidence indicates that the interaction is not mutually exclusive. VTF was shown to bind to UUUUUNU through the N-terminal domain of the large D1 subunit. Furthermore, VTF protects from RNAse A digestion both the 5′ region of the nascent transcript as well as a large central component containing UUUUUNU. The addition of an oligonucleotide containing the 5BrU9 sequence both directly inhibits transcription termination, in vitro and inhibits UV photo crosslinking of VTF to the nascent RNA in the ternary complex. These results support a model in which the availability of the UUUUUNU motif outside of the transcribing RNA polymerase permits binding of both transcription termination factors, VTF and Rap94, to UUUUUNU. The assembly of this termination complex initiates the transcription termination sequence.