The concept of genetic colinearity between the DNA and protein sequences has been found to be violated in many genes during the last ten years. The most obvious example is RNA splicing. Many other examples exist involving unusual mechanisms of transcription and translation. Each of these unusual modes of expression provide molecular biology with new tools to study fundamental genetic processes. We are studying an unusual very efficient ribosomal frameshift which occurs in the Ty elements of the yeast Saccharomyces cerevisiae. The Ty elements of yeast are a dispersed family of 30 related retroviral-like transposons. The elements include two genes, TYA and TYB, which are analogous to gag and pol of retroviruses. TYB is expressed as a fusion to the TYA gene. This fusion could occur by splicing or RNa editing. However since the RNA sequence of the overlap between the two genes is colinear with the DNA only translational models explain TYB expression. We refer to them generically as frameshifting, realizing that mechanism has not been defined. A 14 nucleotide sequence is both necessary and sufficient to promote 20% frameshifting; and that it is the site of the frameshift. This sequence includes no obvious secondary structure, but does include an unusual codon, AGG. This codon is recognized by a rare tRNA encoded by a single genetic locus. It may be that a translational pause induced by the low abundance of the tRNA is required for the frameshift to occur. Many questions remain unanswered about Ty site-specific translational frameshifting. What nucleotides are essential to the process? Would overproduction of the cognate tRNA eliminate frameshifting dependent upon the AGG codon? Could other codons recognized by rare tRNAs substitute? What external factors (ribosomal proteins? translation factors?) regulate the event? Is translational termination required immediately distal to the frameshift site? Frameshifting promoted by the 14 nucleotide minimal site is suppressed near the TYA initiator AUG. This suppression may be caused by the context surrounding the site, though this seems unlikely. Alternatively the early steps in elongation could be different in some way which precludes frameshifting. Frameshifting may operate in a frame-independent way, though most efficient when occurring between the TYA and TYB frames. This result is difficult to reconcile with a model which requires a specific codon-anticodon interaction at the frameshift site. Biochemical analysis can best demonstrate the site of the Ty frameshift. We are purifying the product of a chimeric gene in which a portion of the S. aureus protein A gene is expressed dependent upon the 14 nucleotide minimal site. The sequence of a cyanogen bromide fragment encompassing the frameshift site and protein A fragment will be determined on a gas-phase sequenator. We are also using in vitro translation to demonstrate that an RNA colinear with the DNA can allow TYB expression.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM029480-09
Application #
3277093
Study Section
Genetics Study Section (GEN)
Project Start
1989-08-01
Project End
1994-03-31
Budget Start
1990-04-01
Budget End
1991-03-31
Support Year
9
Fiscal Year
1990
Total Cost
Indirect Cost
Name
University of Maryland Balt CO Campus
Department
Type
Schools of Arts and Sciences
DUNS #
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; 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
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
Vallabhaneni, Haritha; Fan-Minogue, Hua; Bedwell, David M et al. (2009) Connection between stop codon reassignment and frequent use of shifty stop frameshifting. RNA 15:889-97
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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