Nucleic acid binding studies of recombinant HIV-1 nucleocapsid protein /
The nucleocapsid protein (NC) of all animal retroviruses is the major structural protein of the core ribonucleoprotein complex, bound to genomic RNA in mature virions. Biochemical and genetic mutational studies have demonstrated that NC plays integral roles in the structure and replication of all a...
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| Format: | Thesis Book |
| Language: | English |
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[Place of publication not identified] :
[publisher not identified] ;
1995.
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| Online Access: | http://proxy.library.tamu.edu/login?url=http://proquest.umi.com/pqdweb?did=742744791&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | The nucleocapsid protein (NC) of all animal retroviruses is the major structural protein of the core ribonucleoprotein complex, bound to genomic RNA in mature virions. Biochemical and genetic mutational studies have demonstrated that NC plays integral roles in the structure and replication of all animal retroviruses. The subcloning, expression, purification and characterization of defined, recombinant HIV-1 NCs, from an inducible phage T7 RNA polymerase promoter in E. coli. are described. HIV-1 NCl-71 from human immunodeficiency virus type 1 (HIV-1) is a basic protein of 71 an-dno acids, which contains two CYs3MS ZN(II) retroviral-type zinc finger domains. Purified NCl-71 can be reversibly reconstituted with 2 g-at Zn(H) determined by atomic absorption. Ultraviolet circular dichroism (UV-CD), fluorescence spectroscopies and turbidity assays were used to characterize the complexes between highly purified NCs and the model RNA homopolynucleotide poly(A). The binding is complex, showing site-size heterogeneity, slow kinetics and aggregation of the ribonucleoprotein complexes. UV-CD, absorbance and 1H NMR spectroscopies used to study NC complexes with a variety of transfer RNAs appear to show both non-specific binding and similarities between spectral changes that accompany temperature-induced denaturation of the transfer RNAs to NC-induced denaturation. Model studies of the initiation of reverse transcription by HIV-1 reverse transcriptase (RT) and a model 18 nucleotide DNA primer-33 nucleotide RNA template, show that NC modulates the switch from the initiation of reverse transcription step to the processive synthesis step. Studies with defined deletion NCs indicate the central domain is sufficient for the biochemical activities studied, as is the distantly related Mason-Pfizer monkey virus (MPMV) NC. These studies support a non-specific role for NC in some of the biochemical activities attributed to retroviral NCs. The mechanistic implications of these findings on the reported biochemical activities of NC are discussed. |
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| Item Description: | Vita. "Major Subject: Biochemistry". In title, numerals are used. |
| Physical Description: | xiii, 178 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilms Inc. |
| Bibliography: | Includes bibliographical references. |