Structure-function relationships within vaccinia virus protein VP39, a bifunctional protein involved in modification of both mRNA ends /
both mRNA ends. At the 5' end it is an mRNA cap-specific 2' -O-methyltransferase. At the 3' end it is a processivity factor for the virus poly(A) polymerase. Either five or six surface regions of VP39 might be required for its two activities: (1) an S-adenosyl-L-methionine (AdoMet) bi...
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| Format: | Thesis Book |
| Language: | English |
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[Place of publication not identified] :
[publisher not identified] ;
1998.
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| Subjects: | |
| Online Access: | http://proxy.library.tamu.edu/login?url=http://proquest.umi.com/pqdweb?did=737708961&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | both mRNA ends. At the 5' end it is an mRNA cap-specific 2' -O-methyltransferase. At the 3' end it is a processivity factor for the virus poly(A) polymerase. Either five or six surface regions of VP39 might be required for its two activities: (1) an S-adenosyl-L-methionine (AdoMet) binding site', (2) a 5* mRNA cap binding site', (3) a methyltransferase catalytic center; (4) a digitization domain for the poly(A) polymerase catalytic subunit; (5) one or two RNA binding site(s) for VP39's two RNA modifying activities. Structure-function relationships within VP39 were initially analyzed by 'shotgun' mutagenesis, identifying residues involved specifically in 2'-O-methyltransferase activity. Substrate binding assays of methyltransferase-defective mutants identified residues involved in capenhanced RNA binding, AdoMet binding, and possibly also in catalysis. In collaboration with other personnel, the 1.85 x-ray crystal structure of V1339 was determined, along with the binding sites for the methyltransferase cofactor AdoMet and mRNA 5' cap structure, the methyltransferase catalytic center and an adjacent cleft with an apparent RNA binding function. The crystallographic analysis confirmed and extended earlier mutagenesis work, and placed it in a three dimensional context. Surprisingly, 'shotgun' mutagenesis studies shed no light on the surface regions required for the tail elongates activity of VP39. Novel biochemical methods were therefore used to identify two juxtaposed patches of surface sidechairs at the VP55- V1339 digitization interface, using three distinct approaches (in vitro direct digitization assay, protein footprinting analysis and site specific photocrosslinking analysis). In addition, significant conformational changes were also found to occur within V1739 upon digitization with VP55. Having elucidated the minimal number (five) of surface sites for VP39's two RNA modifying functions, it was wondered whether these two activities employed separate RNA binding sites. To this end, 'BlAcore' instrument was used to show that two RNA binding activities may indeed exist in VP39. Thus, VP39 bound to RNA immobilized in the BlAcore cowbell was able to bind a second RNA molecule. |
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| Item Description: | Vita. "Major Subject: Biochemistry". |
| Physical Description: | xii, 150 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilm Inc. |
| Bibliography: | Includes bibliographical references: pages 121-140. |