RNA-protein interactions in mRNA 3'-end polyadenylylation by vaccinia virus poly(A) polymerase /
In vaccinia virus, the mRNA 3' poly (A) tail is formed by a virus-encoded heterodimer of two proteins: VP55, the catalytic subunit of vaccinia poly(A) polymerase, and VP39, the polymerase associated processivity factor. Experiments using (i) the Electrophoretic Mobility Shift Assay (EMSA) in co...
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| Format: | Thesis Book |
| Language: | English |
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[Place of publication not identified] :
[publisher not identified] ;
1999.
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| Subjects: | |
| Online Access: | http://proxy.library.tamu.edu/login?url=http://proquest.umi.com/pqdweb?did=733675491&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | In vaccinia virus, the mRNA 3' poly (A) tail is formed by a virus-encoded heterodimer of two proteins: VP55, the catalytic subunit of vaccinia poly(A) polymerase, and VP39, the polymerase associated processivity factor. Experiments using (i) the Electrophoretic Mobility Shift Assay (EMSA) in combination with various DNA-RNA chimeras, and (ii) a novel ligand selection scheme, demonstrated that VP55 specifically recognizes a rU₂-N₁₅-rU motif (where N denotes any nucleotide) within the primer, while the VP55-VP39 heterodimer recognizes a rU₂-N₂₅-rU motif. Substitution of individual essential uracils of the motif with priding analogs, and EMSA analysis of the resulting oligonucleotides revealed the important functional I groups of the uracil base. A distinctive fingerprint of recognized moieties was observed at each of the three positions, and VP55's ability to discriminate uracil from cytosine was shown to stem largely from a requirement for uracil's protonated N3 nitrogen. Comparing EMSA data with data from a time-course assay measuring the rate of VP55-catalyzed transfer of a labeled chain-terminating adenylate, a 2' OH at the extreme 3' end of the primer was shown to be crucial for catalysis but not for VP55-primer binding stability. In addition, a ribose 2' OH at the -3 position was shown to both stabilize primer binding and stimulate adenylyltransferase activity. Polyadenylylation experiments with a set of sixteen 'tetraU-scanmer' oligonucleotides indicated that VP55 might translocate with respect to the RNA in a manner comparable to inchworming models of transcriptional elongation (Chamberlin, 1995). In a short-range photocrosslinking study employing U[]4-thioU substitutions within the oligonucleotide (dC)₉(rU)₂(dC)₂₅(U)(dC)₈(rC), only the -10-substituted oligonucleotide formed a photocrosslink with VP39, and it did so only in context of the VP55-VP39 heterodimer. R107 was identified as the photocrosslinked residue of V1139 by affinity purification of the crosslinked peptide followed by N-terminal peptide sequencing. This result provided the first structural evidence that VP39 contacts RNA directly in its mRNA 3' end formation function, or that it possesses a polyadenylylation-specific RNA binding site. The latter was shown to be remote from VP39's 5'-end RNA binding 'cleft'. |
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| Item Description: | Vita. "Major Subject: Biochemistry". |
| Physical Description: | xi, 147 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilm Inc. |
| Bibliography: | Includes bibliographical references (leaves 131-143). |