Abstract

The objectives of this proposal are to understand: (1) the relationship between the structure and the many distinctive properties of E. coli initiator tRNA and (2) the molecular mechanisms underlying its specific interactions with components of the translational machinery. An important question is the molecular basis of the highly specific recognition of the tRNA by Met-tRNA transformylase. A combination of structural, biochemical, and genetic approaches will be used to study this. These include (I) NMR spectroscopy to analyze the structure of the tRNA substrate, (ii) crystallography to determine the structure of the protein and the tRNA-protein complex, (iii) investigation of the topology of interaction of the protein with the tRNA using crosslinking experiments, protection experiments and by examining suppressor mutations in the protein that compensate for defects in formylation of mutant tRNAs, and (iv) site-specific mutagenesis to identify amino acids important in the protein for tRNA selection and function. Similar approaches will be used to study interactions of tRNA with other proteins, in particular, in vivo selection and analysis of suppressor mutations in IF2, IF3, or other chromosomal genes. Such work could provide information on interactions between the initiation factors and the initiator tRNA and could lead to identification of new genes involved in translational initiation. Work on identification of intermediates in translation initiation in vivo will continue. Questions of specific interest are: (I) is IF2 a carrier of fMet-tRNA to the ribosome? (ii) Does the 30S ribosome bind first to the initiator tRNA and then to the mRNA or vice versa? (iii) Are the requirements in an initiator tRNA for translational reinitiation the same as for de novo initiation? Finally, the role of the All:U24 base pair unique to eubacterial initiator tRNAs in initiation will be studied along with questions of why introduction of the base pair to an E. coli elongator methionine tRNA prevents accumulation of the tRNA in vivo.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM017151-30
Application #
6018287
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1978-06-01
Project End
2002-06-30
Budget Start
1999-07-01
Budget End
2000-06-30
Support Year
30
Fiscal Year
1999
Total Cost
Indirect Cost

  Institution

Name
Massachusetts Institute of Technology
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
City
Cambridge
State
MA
Country
United States
Zip Code
02139

  Publications

Arguello, Tania; Köhrer, Caroline; RajBhandary, Uttam L et al. (2018) Mitochondrial methionyl N-formylation affects steady-state levels of oxidative phosphorylation complexes and their organization into supercomplexes. J Biol Chem 293:15021-15032
Ghosal, Anubrata; Köhrer, Caroline; Babu, Vignesh M P et al. (2017) C21orf57 is a human homologue of bacterial YbeY proteins. Biochem Biophys Res Commun 484:612-617
Vercruysse, Maarten; Köhrer, Caroline; Shen, Yang et al. (2016) Identification of YbeY-Protein Interactions Involved in 16S rRNA Maturation and Stress Regulation in Escherichia coli. MBio 7:
Niehues, Sven; Bussmann, Julia; Steffes, Georg et al. (2015) Impaired protein translation in Drosophila models for Charcot-Marie-Tooth neuropathy caused by mutant tRNA synthetases. Nat Commun 6:7520
Bhattacharya, Arpita; Köhrer, Caroline; Mandal, Debabrata et al. (2015) Nonsense suppression in archaea. Proc Natl Acad Sci U S A 112:6015-20
Mandal, Debabrata; Köhrer, Caroline; Su, Dan et al. (2014) Identification and codon reading properties of 5-cyanomethyl uridine, a new modified nucleoside found in the anticodon wobble position of mutant haloarchaeal isoleucine tRNAs. RNA 20:177-88
Sinha, Akesh; Köhrer, Caroline; Weber, Michael H W et al. (2014) Biochemical characterization of pathogenic mutations in human mitochondrial methionyl-tRNA formyltransferase. J Biol Chem 289:32729-41
Vercruysse, Maarten; Köhrer, Caroline; Davies, Bryan W et al. (2014) The highly conserved bacterial RNase YbeY is essential in Vibrio cholerae, playing a critical role in virulence, stress regulation, and RNA processing. PLoS Pathog 10:e1004175
Köhrer, Caroline; Mandal, Debabrata; Gaston, Kirk W et al. (2014) Life without tRNAIle-lysidine synthetase: translation of the isoleucine codon AUA in Bacillus subtilis lacking the canonical tRNA2Ile. Nucleic Acids Res 42:1904-15
Jacob, Asha Ivy; Kohrer, Caroline; Davies, Bryan William et al. (2013) Conserved bacterial RNase YbeY plays key roles in 70S ribosome quality control and 16S rRNA maturation. Mol Cell 49:427-38

Showing the most recent 10 out of 36 publications