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Title:Specific Bacteriophage Coat Protein-Rna Interactions
Author(s):Wu, Huey-Nan
Doctoral Committee Chair(s):Uhlenbeck, Olke C.,
Department / Program:Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Chemistry, Biochemistry
Abstract:The coat proteins of the group I bacteriophages translationally repress their replicase genes by binding specifically to a region around the initiation codon called the translational operator. A large number of operator variants have been synthesized by using RNA ligase and transcription of synthetic DNA by T7 RNA polymerase in order to examine the roles of the helical region and the bulged A residue in the specific RNA-R17 coat protein interaction. The affinity between coat protein and each variant was determined by a nitrocellulose filter binding assay. The data of the variants with base pair changes indicated that a stable hairpin loop secondary structure of the operator must be maintained, but any base pairs can be used. 14 different nucleotides were introduced to the bulged A position of these coat protein binding fragments. The data indicated that while functional groups on N$\sp1$, C$\sp2$, C$\sp6$, N$\sp7$ and 2$\sp\prime$OH of the bulged A can be substituted without greatly changing protein binding, bulky substituents cannot be tolerated at these positions.
The sequence and secondary structure of the R17 and fr translational operators differ only at two positions. However, the two coat proteins differ by 17 out of 129 amino acids and have very different isoelectric points. The structural requirements for fr coat protein binding to its operator are virtually identical to that of R17 coat protein. However, fr coat protein binds to nearly every RNA 6 to 14 fold better than R17 coat protein. Part of the tighter binding of the fr coat protein is a result of a more specific interaction to the bulged A residue. RNA fragments containing the operator sequences can initiate the capsid assembly for both coat proteins. Surprisingly, the two coat proteins can form mixed capsid in vitro.
A small ribonucleoprotein particle has been constructed that contains 2 molecules of R17 coat protein dimer and 2 molecules of operator RNA. The formation of this particle was observed by a gel retention assay. It is found that both RNA-RNA and protein-protein interactions were needed to form this complex.
Issue Date:1988
Description:135 p.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1988.
Other Identifier(s):(UMI)AAI8815441
Date Available in IDEALS:2014-12-15
Date Deposited:1988

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