The broad, long-term objective is to understand the molecular basis of the function of the ribosome in protein synthesis. In the near-term the aim is to learn how information is transmitted from one ribosomal domain to another in order to coordinate the partial reactions of protein synthesis. We postulate that signals are conveyed by local or regional changes in rRNA conformation; the result of breaking and remaking tertiary contacts between domains.
In aim 1, we shall test the hypothesis by constructing site-specific, single nucleotide (nt) substitutions in putative tertiary interactions between the sarcin/ricin domain, the peptidyl transferase center, and the GTPase center in E. coli 23S rRNA; we will be guided in this by the atomic structure of the 50S subunit. The intent is to uncover mutations that have a phenotype and, hence, are likely to be important for function. Next, we shall attempt to isolate suppressors of deleterious mutations by constructing a second mutation in the nt partner in the proposed tertiary interaction. We shall also assess the efficiency with which ribosomes with these mutations catalyze the partial reactions of elongation. Finally, we shall analyze the effect of mutations on the conformation of the RNA in the three domains using chemical reagents and Fe (II)-EDTA. The question is whether a mutation in one functional center has an effect on the structure of one or both of the other two centers.
In aim 2 we shall attempt to determine the molecular basis of the cyclic, sequential binding of the elongation factors Tu and G to the ribosome. One possibility, the one we shall investigate first, is that the order is determined by the location on the ribosome of peptidyl-tRNA: that the EF-Tu ternary complex only binds to the ribosome when peptidyl-tRNA is in the P-site, that is to sayin the post translocation state; whereas EF-G.GTP binds when peptidyl-tRNA is in the A-site, i.e., the pretranslocation state. The possibility we espouse is that the position of the peptidyl-tRNA in the A- or P-site, is monitored in the peptidyl transferase center and that a signal generated there is relayed, through a series of tertiary interactions, to the sarcin/ricin domain, initiating in the latter a conformational transition. This is the possibility we shall test using the experimental design and the methods of aim 1.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM033702-18
Application #
6866470
Study Section
Physiological Chemistry Study Section (PC)
Program Officer
Flicker, Paula F
Project Start
1984-09-14
Project End
2009-04-30
Budget Start
2005-05-01
Budget End
2007-04-30
Support Year
18
Fiscal Year
2005
Total Cost
$381,250
Indirect Cost
Name
University of Chicago
Department
Biochemistry
Type
Schools of Medicine
DUNS #
005421136
City
Chicago
State
IL
Country
United States
Zip Code
60637
Yu, Huijun; Chan, Yuen-Ling; Wool, Ira G (2009) The identification of the determinants of the cyclic, sequential binding of elongation factors tu and g to the ribosome. J Mol Biol 386:802-13
Chan, Yuen-Ling; Wool, Ira G (2008) The integrity of the sarcin/ricin domain of 23 S ribosomal RNA is not required for elongation factor-independent peptide synthesis. J Mol Biol 378:12-9
Chan, Yuen-Ling; Dresios, John; Wool, Ira G (2006) A pathway for the transmission of allosteric signals in the ribosome through a network of RNA tertiary interactions. J Mol Biol 355:1014-25
Dresios, John; Chan, Yuen-Ling; Wool, Ira G (2006) Determination of the amino acids in yeast ribosomal protein YS11 essential for the recognition of nucleotides in 18 S ribosomal RNA. J Biol Chem 281:13478-84
Dresios, John; Chan, Yuen-Ling; Wool, Ira G (2005) A determination of the identity elements in yeast 18 S ribosomal RNA for the recognition of ribosomal protein YS11: the role of the kink-turn motif in helix 11. J Mol Biol 345:681-93
Chan, Yuen-Ling; Correll, Carl C; Wool, Ira G (2004) The location and the significance of a cross-link between the sarcin/ricin domain of ribosomal RNA and the elongation factor-G. J Mol Biol 337:263-72
Gluck, Anton; Wool, Ira G (2002) Analysis by systematic deletion of amino acids of the action of the ribotoxin restrictocin. Biochim Biophys Acta 1594:115-26
Dresios, John; Chan, Yuen-Ling; Wool, Ira G (2002) The role of the zinc finger motif and of the residues at the amino terminus in the function of yeast ribosomal protein YL37a. J Mol Biol 316:475-88
Chan, Y L; Sitikov, A S; Wool, I G (2000) The phenotype of mutations of the base-pair C2658.G2663 that closes the tetraloop in the sarcin/ricin domain of Escherichia coli 23 S ribosomal RNA. J Mol Biol 298:795-805
Macbeth, M R; Wool, I G (1999) Characterization of in vitro and in vivo mutations in non-conserved nucleotides in the ribosomal RNA recognition domain for the ribotoxins ricin and sarcin and the translation elongation factors. J Mol Biol 285:567-80

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