Abstract

Chemically-synthesized DNA oligomers less than ten bases long are to be used to probe the functions of various regions of ribosomal RNA in situ. The DNA oligomers will be synthesized so as to be complementary to certain exposed regions of rRNA, hybridized to those regions, assayed for specific binding and then the complex will be assayed to ascertain if the function of the ribosome has been impaired by the presence of the DNA probe. Specific binding of the complementary DNA to a specific rRNA site will be assured by using RNase H to clip the rRNA in the heteroduplex region and then sequencing the resulting fragments for short distances in either direction from the slip. The functional assays to be used will include protein synthesis, tRNA binding to both the P site and the A site, initiation complex formation and mRNA binding assays. It is expected that discrete functions of specific regions of rRNA in the intact ribosome will be determined in this manner.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM035717-03
Application #
3288799
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1986-09-15
Project End
1989-08-31
Budget Start
1988-09-01
Budget End
1989-08-31
Support Year
3
Fiscal Year
1988
Total Cost
Indirect Cost

  Institution

Name
University of Montana
Department
Type
Schools of Arts and Sciences
DUNS #
City
Missoula
State
MT
Country
United States
Zip Code
59812

  Publications

Knight, William; Hill, Walter; Lodmell, J Stephen (2005) Ribosome builder: a software project to simulate the ribosome. Comput Biol Chem 29:163-74
Hennelly, Scott P; Antoun, Ayman; Ehrenberg, Mans et al. (2005) A time-resolved investigation of ribosomal subunit association. J Mol Biol 346:1243-58
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
Brandi, Letizia; Marzi, Stefano; Fabbretti, Attilio et al. (2004) The translation initiation functions of IF2: targets for thiostrepton inhibition. J Mol Biol 335:881-94
Marzi, Stefano; Knight, William; Brandi, Letizia et al. (2003) Ribosomal localization of translation initiation factor IF2. RNA 9:958-69
Bowen, W S; Hill, W E; Lodmell, J S (2001) Comparison of rRNA cleavage by complementary 1,10-phenanthroline-Cu(II)- and EDTA-Fe(II)-derivatized oligonucleotides. Methods 25:344-50
Muth, G W; Hennelly, S P; Hill, W E (2000) Using a targeted chemical nuclease to elucidate conformational changes in the E. coli 30S ribosomal subunit. Biochemistry 39:4068-74
Muth, G W; Thompson, C M; Hill, W E (1999) Cleavage of a 23S rRNA pseudoknot by phenanthroline-Cu(II). Nucleic Acids Res 27:1906-11
Muth, G W; Hennelly, S P; Hill, W E (1999) Positions in the 30S ribosomal subunit proximal to the 790 loop as determined by phenanthroline cleavage. RNA 5:856-64
Bullard, J M; van Waes, M A; Bucklin, D J et al. (1998) Regions of 16S ribosomal RNA proximal to transfer RNA bound at the P-site of Escherichia coli ribosomes. Biochemistry 37:1350-6

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