The aim of this proposal is to study in detail the structure and dynamics of two regions in the E. coli 16S ribosomal RNA, recognizing either S4 protein (greater than 500 bases) or S8 and S15 proteins (approximately 200 bases). The objective of the work is to understand the principles governing the folding and interaction of large RNA molecules. Three complementary approaches will be used: Medium resolution mapping of RNA structures: specific RNA fragments covering the regions of interest will be prepared and probed with structure specific reagents to delineate the RNA secondary structures, regions of major tertiary interactions, and points of contact with bound proteins. The protein binding affinity of fragments will be compared to intact 16S rRNA. Detailed physical studies: once a fairly small fragment of RNA (less than 150 bases) is found to have particularly interesting structural or protein binding features, it will be prepared in quantity for studies by optical and NMR spectroscopy, and its crystallization for x-ray diffraction studies will be attempted. These physical methods are capable of giving a very high resolution picture of the folding, interactions, and dynamics of a limited RNA molecule. The first studies will be with a 112 base fragment containing the S15 binding site and known to have unusually high affinity sites for intercalators. Genetic studies: General schemes will be devised for selecting mutations deficient in a protein - RNA recognition; these will be carried out with S4 protein and the ribosomal and messenger RNAs able to bind it specifically. Mapping of the mutation sites will define the essential RNA and protein features for binding and direct the physical studies to important regions.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Modified Research Career Development Award (K04)
Project #
5K04CA001081-02
Application #
3071645
Study Section
Biophysics and Biophysical Chemistry A Study Section (BBCA)
Project Start
1985-07-01
Project End
1990-06-30
Budget Start
1986-07-01
Budget End
1987-06-30
Support Year
2
Fiscal Year
1986
Total Cost
Indirect Cost
Name
Johns Hopkins University
Department
Type
Schools of Arts and Sciences
DUNS #
045911138
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Tang, C K; Draper, D E (1990) Evidence for allosteric coupling between the ribosome and repressor binding sites of a translationally regulated mRNA. Biochemistry 29:4434-9
Sapag, A; Vartikar, J V; Draper, D E (1990) Dissection of the 16S rRNA binding site for ribosomal protein S4. Biochim Biophys Acta 1050:34-7
Tang, R S; Draper, D E (1990) Bulge loops used to measure the helical twist of RNA in solution. Biochemistry 29:5232-7
Vartikar, J V; Draper, D E (1989) S4-16 S ribosomal RNA complex. Binding constant measurements and specific recognition of a 460-nucleotide region. J Mol Biol 209:221-34
White, S A; Draper, D E (1989) Effects of single-base bulges on intercalator binding to small RNA and DNA hairpins and a ribosomal RNA fragment. Biochemistry 28:1892-7
Tang, C K; Draper, D E (1989) Unusual mRNA pseudoknot structure is recognized by a protein translational repressor. Cell 57:531-6
Ryan, P C; Draper, D E (1989) Thermodynamics of protein-RNA recognition in a highly conserved region of the large-subunit ribosomal RNA. Biochemistry 28:9949-56
Draper, D E (1989) How do proteins recognize specific RNA sites? New clues from autogenously regulated ribosomal proteins. Trends Biochem Sci 14:335-8
Draper, D E; White, S A; Kean, J M (1988) Preparation of specific ribosomal RNA fragments. Methods Enzymol 164:221-37
Draper, D E; Deckman, I C; Vartikar, J V (1988) Physical studies of ribosomal protein-RNA interactions. Methods Enzymol 164:203-20

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