The ribosome is a molecular machine which translates the information encoded in a cell's genome into protein products. It has the twin roles of providing an accurate representation of that information and producing the product rapidly. These roles are fundamentally in conflict since to the extent that translational accuracy increases, the rate of translation decreases. Even so, the ribosome achieves a high degree of accuracy, with an error rate estimated at less than 5 X 10(-4) per codon. How is this very high accuracy achieved? One way of addressing the mechanism of translational accuracy is to consider how specific sequences may perturb it. Programmed frameshift sites are regions of mRNAs which cause efficient changes in reading frame either shifting to the 3' (negative frameshifting) or 5' (positive frameshifting). We would like to understand how one such site which induces +1 frameshifting manipulates the translational apparatus. The retrotransposon Ty3 encodes the product of the POL3 gene as a translational fusion to the upstream GAG3 gene. We have already demonstrated that the event occurs by + l frameshifting within a sequence GCG-AGU-U (shown as codons of GAG3). We have also identified all possible substitutes for the GCG and AGU-U codons. We would like to understand how the frameshift is stimulated. First, we will determine how many 7 nt +1 frameshift sites there are by random oligonucleotide mutagenesis. Second, the tRNA decoding GCG appears to be special in its ability to stimulate frameshifting without itself slipping on the mRNA template. We will attempt to determine what features of this, and other, """"""""P- site"""""""" tRNAs stimulate frameshifting. The """"""""A-site"""""""" tRNA decoding the first +i frame codon, GUU may also be special in driving frameshifting into the +1 frame; we will test this hypothesis by overexpressing and mutagenizing the tRNA. Ty3 frameshifting is stimulated by a downstream """"""""context"""""""", though we don't know how. Some of the hypotheses we will test is that the nascent protein product of the context perturbs frameshifting, or that the context, as RNA, interacts with some element of the translational machinery (elongation factor, ribosomal protein or ribosomal RNA). Finally, we will look for interactions between specific A and P-site tRNAs and other components of the translational machinery to identify trans-acting factors essential to frameshifting. These studies will provide an intellectual basis for understanding the ways in which programmed frameshift sites interact with the translational machinery. The results of these studies will be relevant to our understanding how the ribosome, as a molecular machine, functions to rapidly and accurately decode the genetic information.

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National Institute of General Medical Sciences (NIGMS)
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Genetics Study Section (GEN)
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University of Maryland Balt CO Campus
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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; T├╝kenmez, 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
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Vallabhaneni, Haritha; Farabaugh, Philip J (2009) Accuracy modulating mutations of the ribosomal protein S4-S5 interface do not necessarily destabilize the rps4-rps5 protein-protein interaction. RNA 15:1100-9
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

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