A molecular genetic approach that employs translational suppression is being taken to reveal new ribosomal RNA (rRNA) determinants of specificity in translation and characterize structural determinants of specific functional interactions between rRNA and other translational macromolecules. Long-range goals include finding answers to the following questions: With respect to decoding specificity in the elongation and termination steps of translation in E. coli, what does rRNA do? Which nucleotides, in which of the three rRNAs, do it? Which nucleotides are involved in specific functional interactions with transfer RNAs (tRNAs) or with external insults such as virus-encoded antisense RNAs? The specific aims are: (1) To select for new kinds of rRNA translational suppressors, determine their mutational changes, and characterize their altered translational functions in vivo. (2) To select for rRNA mutants that interact specifically, in decoding, with one of two particular parts of a tRNA molecule, the acceptor stem or anticodon loop, determine their DNA sequence changes, and study the precise interactions with that part of the tRNA. (3) To characterize further the suppressor and inhibitory actions of the RNA encoded by a cloned phage lambda bar segment, as to its apparent involvement in peptide chain termination and its possible interaction or co-functioning with the RNA of the small subunit of the ribosome. For the most part, cloned rRNA genes will be subjected to random mutagenesis in vivo. E. coli cells containing well-characterized trpA mutations will be transformed with the mutagenized plasmid and spread on media to select simultaneously for plasmic acquisition and suppression of the trpA mutations. Some recipient cells will also contain a suppressor tRNA that in the presence of wild-type rRNA does not suppress the particular trpA mutation. Information from these studies could lead to development of therapeutic procedures that discriminate against detrimental viruses or bacteria that depend upon specialized translational events for their propagation. It will also be most important for attempts to accurately engineer and produce medically and industrially important proteins.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM021499-23
Application #
2900498
Study Section
Special Emphasis Panel (ZRG5-MBC-1 (01))
Project Start
1978-03-01
Project End
2001-03-31
Budget Start
1999-04-01
Budget End
2000-03-31
Support Year
23
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of Texas MD Anderson Cancer Center
Department
Genetics
Type
Other Domestic Higher Education
DUNS #
001910777
City
Houston
State
TX
Country
United States
Zip Code
77030
Bouakaz, Lamine; Bouakaz, Elli; Murgola, Emanuel J et al. (2006) The role of ribosomal protein L11 in class I release factor-mediated translation termination and translational accuracy. J Biol Chem 281:4548-56
Bowen, William S; Van Dyke, Natalya; Murgola, Emanuel J et al. (2005) Interaction of thiostrepton and elongation factor-G with the ribosomal protein L11-binding domain. J Biol Chem 280:2934-43
Van Dyke, Natalya; Murgola, Emanuel J (2003) Site of functional interaction of release factor 1 with the ribosome. J Mol Biol 330:9-13
Xu, Wenbing; Pagel, Frances T; Murgola, Emanuel J (2002) Mutations in the GTPase center of Escherichia coli 23S rRNA indicate release factor 2-interactive sites. J Bacteriol 184:1200-3
Van Dyke, Natalya; Xu, Wenbing; Murgola, Emanuel J (2002) Limitation of ribosomal protein L11 availability in vivo affects translation termination. J Mol Biol 319:329-39
Arkov, Alexey L; Hedenstierna, Klas O F; Murgola, Emanuel J (2002) Mutational eidence for a functional connection between two domains of 23S rRNA in translation termination. J Bacteriol 184:5052-7
Hedenstierna, K O; Murgola, E J (2001) Targeting random mutations to regions that are not flanked by existing restriction sites. Biotechniques 30:482-4, 486
Arkov, A L; Freistroffer, D V; Pavlov, M Y et al. (2000) Mutations in conserved regions of ribosomal RNAs decrease the productive association of peptide-chain release factors with the ribosome during translation termination. Biochimie 82:671-82
Chernyaeva, N S; Murgola, E J (2000) Covariance of complementary rRNA loop nucleotides does not necessarily represent functional pseudoknot formation in vivo. J Bacteriol 182:5671-5
Hedenstierna, K O; Siefert, J L; Fox, G E et al. (2000) Co-conservation of rRNA tetraloop sequences and helix length suggests involvement of the tetraloops in higher-order interactions. Biochimie 82:221-7

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