The process of protein synthesis on the ribosome is among the oldest of biological mechanisms, having evolved probably in the RNA world before the first cells, The ribosome has evolved an elaborate structure, the essential parts conserved in all domains of life, Understanding the mechanism by which the ribosome faithfully translates mRNAs into proteins has become both more important and more tractable with the availability of multiple molecular structures of both subunits of bacterial ribosomes with various ligands, Our laboratory for many years has been interested in the problem of how mRNA sequences interact with the ribosome to cause programmed errors, We have mainly studied the mechanism of programmed translational frameshifting in which particular sites in mRNAs stimulate translational errors at rates several orders of magnitude greater than at random positions in mRNAs, Our work and that of others revealed that programmed frameshift sites manipulate the decoding center of the ribosome to exacerbate errors. More recently, we have begun to study the mechanism of missense errors, Our study has shown that errors vary widely by type of missense error (first, second or wobble position mispairing) and according to the tRNA isoacceptors involved in decoding the codon that is the site of the error. The major conclusion of our work is that wobble errors commonly occur, tllOugh they are not universal, and that other types of errors occur when the competing cognate tRNA is in insufficient supply to preclude decoding by the errant tRNA, We propose to further characterize the phenomenology of missense errors by creating a set of error-reporter constructs based on the E, coli lacZ and the Photinus (firefly) luciferase genes. Venki Ramakrishnan has proposed that a major part of the accuracy mechanism requiring the disruption ofa protein.protein interaction between ribosomal proteins S4 (rpS4) and rpS5, which allows the ribosome to shift into a "closed" conformation that traps the aminoacyl-tRNA in the A site and allows exit of EF-l A.GDP, The model is based on the existence of mutants targeting the interface that are proposed to destabilize it and cause increased errors, Our preliminary data are inconsistent with this hypothesis, We propose to further test the hypothesis by identifying novel mutations that alter accuracy, either in the rRNA or ribosomal proteins, The process of protein synthesis on the ribosome is among the oldest of biological mechanisms, having evolved probably in the RNA world before the first cells, The ribosome has evolved an elaborate structure, the essential parts conserved in all domains of life, Understanding the mechanism by which the ribosome faithfully translates mRNAs into proteins has become both more important and more tractable with the availability of multiple molecular structures of both subunits of bacterial ribosomes with various ligands, Our laboratory for many years has been interested in the problem of how mRNA sequences interact with the ribosome to cause programmed errors, We have mainly studied the mechanism of programmed translational frameshifting in which particular sites in mRNAs stimulate translational errors at rates several orders of magnitude greater than at random positions in mRNAs, Our work and that of others revealed that programmed frameshift sites manipulate the decoding center of the ribosome to exacerbate errors. More recently, we have begun to study the mechanism of missense errors, Our study has shown that errors vary widely by type of missense error (first, second or wobble position mispairing) and according to the tRNA isoacceptors involved in decoding the codon that is the site of the error. The conclusion of our work is that wobble errors commonly occur, tllOugh they are not universal, and that other types of errors occur when the competing

Public Health Relevance

The process of protein synthesis is highly accurate yet errors do occur, though less than once in 10,000 amino acids incorporated. This proposal will determine the spectrum of these errors (what types of errors and how often they occur) as well as genetically determine what parts of the protein synthetic machine, the ribosome, are responsible for keeping them to a minimum. An understanding of these errors is important because the protein synthetic mechanism is arguably the oldest system at work in the cell. The process is frequently the target of diseases including cancer and AIDS. It is also frequently the target of antibiotics making an understanding of the process essential to develop drugs to replace those no longer useful clinically because of resistance.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM029480-26
Application #
7896489
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Bender, Michael T
Project Start
1989-08-01
Project End
2012-06-30
Budget Start
2010-07-01
Budget End
2012-06-30
Support Year
26
Fiscal Year
2010
Total Cost
$327,584
Indirect Cost
Name
University of Maryland Balt CO Campus
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
061364808
City
Baltimore
State
MD
Country
United States
Zip Code
21250
Manickam, Nandini; Joshi, Kartikeya; Bhatt, Monika J et al. (2016) Effects of tRNA modification on translational accuracy depend on intrinsic codon-anticodon strength. Nucleic Acids Res 44:1871-81
Nord, Stefan; Bhatt, Monika J; Tükenmez, Hasan et al. (2015) Mutations of ribosomal protein S5 suppress a defect in late-30S ribosomal subunit biogenesis caused by lack of the RbfA biogenesis factor. RNA 21:1454-68
Manickam, Nandini; Nag, Nabanita; Abbasi, Aleeza et al. (2014) Studies of translational misreading in vivo show that the ribosome very efficiently discriminates against most potential errors. RNA 20:9-15
Turkel, Sezai; Kaplan, Guliz; Farabaugh, Philip J (2011) Glucose signalling pathway controls the programmed ribosomal frameshift efficiency in retroviral-like element Ty3 in Saccharomyces cerevisiae. Yeast 28:799-808
Kramer, Emily B; Vallabhaneni, Haritha; Mayer, Lauren M et al. (2010) A comprehensive analysis of translational missense errors in the yeast Saccharomyces cerevisiae. RNA 16:1797-808
Kramer, Emily B; Farabaugh, Philip J (2007) The frequency of translational misreading errors in E. coli is largely determined by tRNA competition. RNA 13:87-96
Guarraia, Carla; Norris, Laura; Raman, Ana et al. (2007) Saturation mutagenesis of a +1 programmed frameshift-inducing mRNA sequence derived from a yeast retrotransposon. RNA 13:1940-7
Taliaferro, Dwayne L; Farabaugh, Philip J (2007) Testing constraints on rRNA bases that make nonsequence-specific contacts with the codon-anticodon complex in the ribosomal A site. RNA 13:1279-86
Taliaferro, Dwayne; Farabaugh, Philip J (2007) An mRNA sequence derived from the yeast EST3 gene stimulates programmed +1 translational frameshifting. RNA 13:606-13
Raman, Ana; Guarraia, Carla; Taliaferro, Dwayne et al. (2006) An mRNA sequence derived from a programmed frameshifting signal decreases codon discrimination during translation initiation. RNA 12:1154-60

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