Protein-Protein and Protein-Rna Interactions in the in Vitro Assembly of the R17 Bacteriophage
Beckett, Dorothy
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Permalink
https://hdl.handle.net/2142/70555
Description
Title
Protein-Protein and Protein-Rna Interactions in the in Vitro Assembly of the R17 Bacteriophage
Author(s)
Beckett, Dorothy
Issue Date
1986
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Biochemistry
Abstract
The R17 bacteriophage is a positive single stranded RNA virus that specifically infects male strains of E. coli. The role of RNA protein interactions in the in vitro assembly of the virus is examined in order to determine both what features of the RNA are important for its selective packaging by R17 coat protein and how the polymerization of the coat protein into the T = 3 structure is related its interaction with the RNA.
The oligomeric state of the R17 coat protein in the unassembled state was shown to be dimeric. The signal for selective packaging of R17 RNA by its coat protein has been proposed to be the translational operator of the phage encoded replicase gene. The synthesis and characterization of the coat protein binding properties of a fragment that is a functional analog of the translational operator sequence is described. A single complex forms between a coat protein dimer and the translational operator fragment. At sufficiently high fragment and coat protein concentrations T = 3 capsids form which, according to kinetic and stoichiometric studies, result from polymerization of the fragment-dimer complex. In the same conditions coat protein alone remains dimeric. Binding of the translational operator fragment induces coat protein polymerization into T = 3 shells.
The translational operator sequence constitutes only a small fraction of the entire R17 genome and sequences outside of the operator demonstrate no detectable affinity for the coat protein dimer and can be considered non-specific. Despite their lack of measurable affinity for the coat protein dimer non-specific RNAs also induce polymerization of R17 coat protein into T = 3 shells. Measurement of coat protein concentration dependence of assembly in the presence of several non-specific RNAs indicates that the length of the RNA is important in determining the efficiency with which assembly occurs. Since selective packaging has been proposed to depend on the presence of a single copy of the translational operator sequence in the R17 genome the effect of introduction of this sequence into a long nonspecific RNA on the overall assembly process is examined. Comparison of assembly in the presence of two RNAs, identical except for the presence of a single copy of the operator sequence in one, indicate that introduction of the specific site confers a small advantage on an RNA in assembly. The majority of the free energy in the entire assembly process derives, however, from coupling of coat protein polymerization to interaction of the coat protein with non-specific RNA sequences. (Abstract shortened with permission of author.)
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