Analysis of the Roles of tRNA Structure, Ribosomal Protein L9, and the Bacteriophage T4 gene 60 Bypassing Signals during Ribosome Slippage on mRNA Alan J. Herr, Chad C. Nelson, Norma M. Wills, Raymond F. Gesteland and John F. Atkins* Department of Human Genetics, University of Utah 2030 E 15N, RM 7410, Salt Lake City, UT 84112- 5330, USA A 50-nucleotide coding gap divides bacteriophage T4 gene 60 into two open reading frames. In response to cis-acting stimulatory signals encrypted in the mRNA, the anticodon of the ribosome-bound peptidyl tRNA dissociates from a GGA codon at the end of the ®rst open reading frame and pairs with a GGA codon 47 nucleotides downstream just before the second open reading frame. Mutations affecting ribosomal pro- tein L9 or tRNA Gly 2 , the tRNA that decodes GGA, alter the ef®ciency of bypassing. To understand the mechanism of ribosome slippage, this work analyzes the in¯uence of these bypassing signals and mutant trans- lational components on 1 frameshifting at G GGA and hopping over a stop codon immediately ¯anked by two GGA glycine codons (stop-hop- ping). Mutant variants of tRNA Gly 2 that impair bypassing mediate stop- hopping with unexpected landing speci®cities, suggesting that these var- iants are defective in ribosomal P-site codon-anticodon pairing. In a direct competition between 1 frameshifting and stop-hopping, the absence of L9 promotes stop-hopping at the expense of 1 frameshifting without substantially impairing the ability of mutant tRNA Gly 2 variants to re-pair with the mRNA by sub-optimal pairing. These observations suggest that L9 defects may stimulate ribosome slippage by enhancing mRNA movement through the ribosome rather than by inducing an extended pause in translation or by destabilizing P-site pairing. Two of the bypassing signals, a cis-acting nascent peptide encoded by the ®rst open reading frame and a stemloop signal located in the 5 0 por- tion of the coding gap, stimulate peptidyl-tRNA slippage independently of the rest of the gene 60 context. Evidence is presented suggesting that the nascent peptide signal may stimulate bypassing by destabilizing P-site pairing. # 2001 Academic Press Keywords: hopping; bypassing; ribosomal protein L9; nascent peptide; tRNA Gly 2 *Corresponding author Introduction Reading-frame maintenance during translation requires stable base-pairing between transfer RNA (tRNA) and messenger RNA (mRNA) in the ribo- somal P-site. The ribosome appears to stabilize P-site tRNA-mRNA pairing through extensive contacts to the peptidyl-tRNA-mRNA complex. 1±3 Despite this investment in maintaining P-site pair- ing, error slippage of the peptidyl-tRNA occurs at low levels at certain sites in the mRNA with vari- able ef®ciency. When the peptidyl-tRNA detaches Present address: A. J. Herr, The Sainsbury Laboratory, John Innes Centre, Colney Lane, Norwich NR4 7UH, UK. Abbreviations used: GST, glutathione S-transferase; MBP, maltose-binding protein; 6His, six histidine residues; SD, Shine-Dalgarno; trxA, thioredoxin; tet r , tetracycline resistance. E-mail address of the corresponding author: john.atkins@genetics.utah.edu doi:10.1006/jmbi.2001.4717 available online at http://www.idealibrary.com on J. Mol. Biol. (2001) 309, 1029±1048 0022-2836/01/051029±20 $35.00/0 # 2001 Academic Press