Analysis of site-specific interaction parameters in protein-DNA complexes.
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Chromatography, Gel↗
Biomedical subjects
D Beckett
Publications and source records attributed to D Beckett.
Quantitative study of protein association at picomolar concentrations: the lambda phage cl repressor.
A method has been developed for radiolabeling the lambda cl repressor to a specific activity sufficiently high to permit accurate quantitation of the protein in the picomolar range of concentration. Procedures are described whereby the labeled protein can be used for accurate quantitative study of the energetics of repressor assembly by large zone analytical gel chromatography. This methodology is applicable to other systems in which the stoichiometry and energetics of tightly associating DNA binding proteins are currently difficult to measure.
Chromatography, Gel↗
Ribonucleoprotein complexes of R17 coat protein and a translational operator analog.
The coat protein of the simple spherical (triangulation no. T = 3) RNA coliphage R17 protects the genomic RNA in the virus particle and acts as a translational repressor of the phage-encoded replicase gene. It has been suggested that these two functions are related and that the translational repression complex serves as a nucleation complex for subsequent assembly of the bacteriophage. We have used a translational operation fragment to examine the relationship between formation of the translational repression complex and the assembly of the protein into T = 3 capsids. In vitro analysis of the aggregation properties of R17 coat protein reveals that binding of the translational operator fragment to the protein dimer triggers polymerization of the protein into T = 3 capsids of well-defined composition. The data further implicate the translational operator in nucleation of assembly and suggest a possible physical-chemical basis of the nucleation step.
Base Sequence↗
Roles of operator and non-operator RNA sequences in bacteriophage R17 capsid assembly.
In order to understand the role of sequences other than the translational operator on bacteriophage R17 assembly, in vitro capsid assembly was studied with R17 coat protein and a variety of RNAs. For a series of RNA oligomers of the same chain length, sequences that bind coat protein dimer with a lower affinity require higher concentrations of RNA and protein for assembly. Among a series of non-specific RNA molecules of differing lengths, lower protein and RNA concentrations are required for assembly of capsids containing longer RNAs. For RNA molecules of any length, the presence of a single high-affinity translational operator sequence lowered the concentration requirements for capsid assembly. However, the advantage for encapsidation provided by the operator sequence is small for large RNA molecules. The experiments indicate that in the overall assembly process the interaction of coat protein with non-specific sequences is at least as important as its interaction with the specific translational operator sequence. In light of the data, a mechanism of achieving selective packaging of the R17 genomic RNA in vivo is discussed.
Bacteriophages↗