Title

Identification and characterization of cis-acting signals in the genome of vesicular stomatitis virus that control polyadenylation, transcription termination, and transcription reinitiation

Date of Award

1999

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Microbiology and Immunology

First Committee Member

Asit K. Pattnaik, Committee Chair

Abstract

Each of the gene junctions of VSV contains a 23-nucleotide long conserved sequence, 3'-AUACU7(G/C)AUUGUCnnUAG-5 ' where n is nonconserved nucleotide. It has long been suspected that these conserved sequences mediate various transcription activities such as polyadenylation, transcription termination, and transcription reinitiation by the polymerase at the gene junctions. To investigate the role of the sequences in these processes, we have established a minigenome-expression system in which transcriptionally- and replicationally-active minigenomic RNAs are synthesized. By introducing mutations at key sequences within the gene junctions of these minigenomes, we have investigated the role of the conserved sequence elements at the gene junctions in signaling polymerase activity.These studies provide evidence that the putative polyadenylation signal, AUACU7, is necessary and sufficient to induce both polyadenylation and transcription termination of the upstream RNA. In addition, further characterization of the sequence shows that the sequence element can tolerate nucleotide changes in the first three positions as well as an increase in the length of the uridine stretch up to ten residues without adversely affecting polyadenylation and transcription termination. On the other hand substitutions at the fourth base or decreasing the length of the uridine tract cannot be tolerated.Results from studies on the role of sequences at the leader-N gene junction in transcription reinitiation suggest that both conserved UUGUC and UAG sequence elements at the beginning of the N gene are required for efficient transcription reinitiation. In addition, mutant templates exhibiting decreased transcription reinitiation from the authentic site generate a smaller transcript that is reinitiated at a cryptic site located within the gene. Primer extension analysis shows that the smaller transcript is reinitiated from a sequence UUGUCGAUG located 220 nucleotides downstream of the normal reinitiation site. Changes made within this downstream cryptic reinitiation site provide additional evidence for reinitiation at this site. The characterization of the sequence elements in the VSV genome signaling polyadenylation, transcription termination, and transcription reinitiation is the first step towards understanding the mechanisms by which VSV and other negative-stranded RNA virus genomes are transcribed.

Keywords

Biology, Molecular; Biology, Genetics; Biology, Microbiology

Link to Full Text

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