The goal of the proposed research is to understand the mechanisms by which ribosomal proteins recognize specific RNA sites, and the influence protein binding may have on RNA structure. Thermodynamic, chemical modification, and genetic techniques will be used to study three protein-RNA interaction systems. Using an in vitro transcription system, sets of rRNA or mRNA fragments differing in length or sequence will be prepared, and their ability to specifically bind proteins tested. Physical studies of the RNA fragments and the RNA-protein complexes will show whether protein binding influences the RNA conformation. The three systems to be studied are: i) an extended 97 base RNA hairpin containing the recognition site for protein S15. This RNA is already known to contain unusually high affinity sites for ethidium intercalation, and to undergo a distinct conformational change upon ethidium binding. Whether protein binding is coupled to this conformational change will be investigated. ii) The protein S4 recognizes both the 16S rRNA, and the mRNA coding for S4 and several other ribosommal proteins. Regions of the mRNA required for S4 binding have already been defined. Further work will delineate the specific nucleotides required for recognition in each RNA, and compare the thermodynamics of binding to each RNA. Genetic systems capable of selecting mutants in the rRNA, mRNA, or protein defective in recognition will be used to help detect important recognition features. iii) The proteins L10 and L11 bind cooperatively to a limited, about 100 base region of the 23S rRNA. This region of rRNA is highly conserved and important for the GTPase activity of translating ribosomes. The protein binding sites, mechanism of cooperatively, and possibility of reconstituting GTPase activity in a small complex of proteins and rRNA will be investigated.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM029048-10
Application #
3276518
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Project Start
1981-04-01
Project End
1992-03-31
Budget Start
1990-04-01
Budget End
1991-03-31
Support Year
10
Fiscal Year
1990
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
Bausch, Sarae L; Poliakova, Ekaterina; Draper, David E (2005) Interactions of the N-terminal domain of ribosomal protein L11 with thiostrepton and rRNA. J Biol Chem 280:29956-63
Maeder, Corina; Draper, David E (2005) A small protein unique to bacteria organizes rRNA tertiary structure over an extensive region of the 50 S ribosomal subunit. J Mol Biol 354:436-46
Conn, Graeme L; Gittis, Apostolos G; Lattman, Eaton E et al. (2002) A compact RNA tertiary structure contains a buried backbone-K+ complex. J Mol Biol 318:963-73
Shiman, R; Draper, D E (2000) Stabilization of RNA tertiary structure by monovalent cations. J Mol Biol 302:79-91
GuhaThakurta, D; Draper, D E (1999) Protein-RNA sequence covariation in a ribosomal protein-rRNA complex. Biochemistry 38:3633-40
Conn, G L; Gutell, R R; Draper, D E (1998) A functional ribosomal RNA tertiary structure involves a base triple interaction. Biochemistry 37:11980-8
Rogers, M J; Bukhman, Y V; McCutchan, T F et al. (1997) Interaction of thiostrepton with an RNA fragment derived from the plastid-encoded ribosomal RNA of the malaria parasite. RNA 3:815-20
Sapag, A; Draper, D E (1997) In vitro evolution used to define a protein recognition site within a large RNA domain. Bioorg Med Chem 5:1097-105
Gluick, T C; Gerstner, R B; Draper, D E (1997) Effects of Mg2+, K+, and H+ on an equilibrium between alternative conformations of an RNA pseudoknot. J Mol Biol 270:451-63
Wang, Y X; Huang, S; Draper, D E (1996) Structure of a U.U pair within a conserved ribosomal RNA hairpin. Nucleic Acids Res 24:2666-72

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