CONTROL OF GENE EXPRESSION BY TRANSLATIONAL RECODING By JONATHAN D DINMAN Department of Cell Biology and Molecular Genetics University of Maryland College Park Maryland USA I II III IV V VI Introduction Programmed 1 Ribosomal Frameshifting A Introduction and History B PRF in Viruses The Golden Mean and Possible Therapeutic Applications C Computational Identification of 1 PRF Signals Genomic Frameshifting D 1 PRF in Cellular mRNAs mRNA Destabilization Elements and Regulation of Gene Expression Programmed 1 Ribosomal Frameshifting A History of 1 PRF Retrotransposable Elements B Cellular 1 PRF Selenocysteine and Pyrrolysine The 21st and 22nd Amino Acids A Selenocysteine B Pyrrolysine Termination Codon Readthrough A History In Viruses B Termination Suppression in Cellular Genes C Therapeutic Applications Summary References 130 131 131 132 133 135 136 136 137 139 139 140 141 141 141 142 142 143 Abstract Like all rules even the genetic code has exceptions these are generically classified as translational recoding Almost every conceivable mode of recoding has been documented including signals that redefine translational reading frame and codon assignation While first described in viruses it is becoming clear that sequences that program elongating ribosomes to shift translational reading frame are widely used by organisms in all domains of life thus expanding both the coding capacity of genomes and the modes through which gene expression can be regulated at the posttranscriptional level Instances of programmed ribosomal ADVANCES IN PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY Vol 86 DOI 10 1016 B978 0 12 386497 0 00004 9 129 Copyright 2012 Elsevier Inc All rights reserved 130 DINMAN frameshifting and stop codon reassignment are opening up new avenues for treatment of numerous inborn errors of metabolism The implications of these findings on human health are only beginning to emerge I Introduction Many years ago I took a Japanese colleague on a walking tour of Manhattan starting in SoHo and ending up at Rockefeller Center Having come from Tokyo my guest was not so much overwhelmed by the crowds as he was perplexed by the fact that New Yorkers tended to cross the street against the light By the time we had reached Times Square he turned to me and said I understand don t walk means run The point is that sometimes rules can be safely and advantageously broken Indeed exceptions to the rules provide the contrasting points of reference that enable definition of the rules themselves Every hero needs a foil there could be no Hamlet without Laertes Consider the Central Dogma of molecular biology information flows from DNA to RNA to protein The discovery of reverse transcriptase shattered this rule and opened up broad new vistas in both our understanding and ability to manipulate biological systems Not insignificantly this discovery garnered Nobel Prizes for Drs Temin and Varmus demonstrating that challenging the status quo can sometimes be very rewarding The genetic code is another set of rules defining how amino acids are encoded in nucleic acid sequences Given that four nucleotide bases must encode 20 amino acids Nirenberg surmised that a minimum of three bases had to be used to encode one amino acid Based on this and using defined templates in well defined in vitro translation assay systems he deciphered the genetic code and demonstrated that it is the same in Escherichia coli Xenopus laevis and guinea pig tissues a universally conserved genetic code composed of nucleoside triplets Given the organization of genetic information into triplet codons it became apparent that ribosomes the universally conserved protein synthetic machinery have to accurately recognize and decode bases three at a time in order to accurately translate the genetic information contained in mRNAs into proteins These findings laid the foundation for investigations designed to answer the fundamental questions of translational accuracy and reading frame maintenance As described elsewhere the discoveries of exceptions to the genetic code generically termed translational recoding have helped to TRANSLATIONAL RECODING AND GENE EXPRESSION 131 address these questions and have opened new vistas in our understanding of many other biological questions reviewed in Atkins and Gesteland 2010 This chapter focuses on recent efforts to identify translational recoding signals in cellular genomes and their known and hypothetical roles as cis acting elements in controlling gene expression II Programmed 1 Ribosomal Frameshifting A Introduction and History Programmed ribosomal frameshifting PRF is a translational recoding phenomenon historically associated with viruses and retrotransposons PRF signals are cis acting elements embedded in mRNAs that stochastically redirect translating ribosomes into a new reading frame i e by 1 or 1 nucleotide In the typical viral context a PRF signal allows ribosomes to bypass the 0 frame encoded in frame stop codon and continue synthesis of a C terminally extended fusion protein The most well defined 1 PRF phenomena are directed by an mRNA sequence motif composed of three important elements a slippery site composed of seven nucleotides where the translational shift in reading frame actually takes place a short spacer sequence of usually 12 nucleotides and a downstream stimulatory structure typically an mRNA pseudoknot In eukaryotic viruses the slippery site has the heptameric motif N NNW WWH where the incoming reading frame is indicated and N any three identical nucleotides W AAA or UUU and N6 G Harger et al 2002 Current models posit that aminoacyl aa and peptidyl tRNAs are positioned on this sequence while the ribosome pauses at the downstream secondary structure Lopinski et al 2000 Kontos et al 2001 Plant et al 2003 Plant and Dinman 2005 The nature of the slippery sequence enables re pairing of the non wobble bases of both the aa and peptidyl tRNAs with the 1 frame codons While it is generally accepted that mRNA pseudoknots are the most common type of downstream stimulatory structures other mRNA structures are capable of filling this role as well Kollmus et al 1996 Baril et al 2003 Generally it is thought that the essential function of the stimulatory structure is to provide an energetic barrier to a translating ribosome and to position it over the slippery site A number of models have been presented to predict at which point during the course of the 132 DINMAN translation elongation cycle 1 PRF occurs reviewed