Posttranscriptional regulation of baculovirus late and very late genes /
Baculoviruses late genes are transcribed by a viral-encoded RNA polymerase that is a complex of four subunits, LEF-4, LEF-8, LEF-9 and p47. The viral mRNAS, including late transcripts, are capped at the 5' ends with a typical methyl-7-guanosine structure and polyadenylated at the 3' end. T...
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| Format: | Thesis Book |
| Language: | English |
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[Place of publication not identified] :
[publisher not identified] ;
2000.
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| Online Access: | http://proxy.library.tamu.edu/login?url=http://proquest.umi.com/pqdweb?did=731990141&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | Baculoviruses late genes are transcribed by a viral-encoded RNA polymerase that is a complex of four subunits, LEF-4, LEF-8, LEF-9 and p47. The viral mRNAS, including late transcripts, are capped at the 5' ends with a typical methyl-7-guanosine structure and polyadenylated at the 3' end. This poses an interesting question regarding the mechanism of posttranscriptional regulation of baculovirus late and very late mRNAs. We found that purified viral RNA polymerase had guanylyltransferase, NTPase and RNA triphosphatase activities that were exactly copurified with transcription activity. Amino acid sequence analysis revealed guanylyltransferase and RNA triphosphatase motifs in the LEF-4 subunit of RNA polymerase, suggesting that LEF-4 was responsible for these activities. In vitro enzymatic assays with purified LEF-4 single subunit and mutational analyses of conserved residues confirmed that LEF-4 is a mRNA capping enzyme. NTPase and RNA triphosphatase functions map to the N-terminal portion of LEF-4, while the C-terminal region has guanylyltransferase activity. We also found that a temperature sensitive mutation, which has a substitution of phenylalanine for leucine at position 105, had no significant effect on the ATPase or guanylyltransferase activity of LEF-4 but resulted in a modest decrease in mRNA triphosphatase activity. A synthetic globes sequence was cloned into the polyhedron C-free cassette that is used as a transcription template. In vitro transcription assays, nucleotide chase experiments, transcription time course, and northern blotting analyses indicated that the RNA transcripts made by purified viral RNA polymerase were terminated and polyadenylated. RNA 3' RACE and cDNA sequence analyses showed that termination occurred at two T-rich regions in the globin sequence. Sequence mutation analysis indicated that the poly (A) signal is not essential for transcription termination of viral RNA polymerase. The transcription termination of baculovirus RNA polymerase is independent of ATP hydrolysis. |
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| Item Description: | Vita. "Major Subject: Biochemistry". |
| Physical Description: | xv, 142 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilm Inc. |
| Bibliography: | Includes bibliographical references (leaves 119-140). |