Chapter 40 : Factor-Mediated Termination of Protein Synthesis: a Welcome Return to the Mainstream of Translation

Sequence alignment of the region encompassing the RF type-specific sequences (positions 190 to 217) of selected prokaryotic RFs. Conserved (black boxes) and similar (light gray boxes with black text) residues between all RFs and conserved residues specific for each RF type (dark gray boxes with white text) are presented. Mutations (a to f) clustered within this region are documented in Table 1 . The abbreviations are defined in the legend to Fig. 2, except the following: Bfi, Bacillus firmus; Hpy, Helicobacter pylori; Bbu, Borrelia burgdorferi; and Tth, Thermus thermophilus.

Similarity between prokaryotic and organellar IF1s and class I RFs. The two types of class I RFs, RF1 and RF2, have been considered as separate families, since their stop codon specificities (UAA and UAG for RF1; UAA and UGA for RF2) suggest that significant deviations may exist in their interactions with mRNA and the decoding site. (A) Positions of amino acids identical among all currently available prokaryotic and organellar RFs (all RFs), RF1s (RF1), RF2s (RF2), and IF1s (IF1) are indicated by vertical bars. The alignments were accomplished with the PILEUP program (GCG package, University of Wisconsin Genetics Computer Group). (B) Alignment of distantly related members of the IF1 and RF families. IF1 and RF amino acid residues that are identical or functionally related in at least three IF1s and three RF1s or two RF2s are indicated. The following sets of amino acids are considered to be functionally related: A and G; S, T, and C; L, I, V, M, and F; F, Y, W, and H; K and R; D and E; D and N; and E and Q. Abbreviations: Bsu, B. subtilis; Cbu, Coxiella burnetii; Ctr, Chlamydia trachomatis; Eco, E. coli; Hin, Haemophylus influenzae; Lla, Lactococcus lactis; Mbo, Mycobacterium bovis; Mca, Mycoplasma capricolum; Mge, Mycoplasma genitalium; Mle, Mycobacterium leprae; Mpn, Mycoplasma pneumoniae; Mpo, Marchantia polymorpha; Mtu, Mycobacterium tumefaciens. cZma, cSol, cNta, cOsa, cEvi, and cPth are chloroplast IF1s of Zea mays, Spinacia oleracea, Nicotiana tabacum, Oryza sativa, Epifagus virginiana, and Pinus thunbergii, respectively.

Dot blot of E. coli IF1 against RF2 and RF3 against EFG. The full sequences of IF1 (positions 1 to 71) and RF2 (1 to 366) (A) and RF3 (1 to 527) and EF-G (1 to 703) (B) were compared to determine regions of similarity by using the Genetics Computer Group programs Compare and Dotplot. Dotplot was run at a density of 306.67. The alignments were performed with a stringency value of 15.

Predicted structure of RF3 based on threading analyses with EF-G. The predicted structure of RF3 is compared with the known three-dimensional structure of EF-G. Threading of RF3 sequences onto EF-G was performed with Swiss-Model ( Guex and Peitsch, 1997 ).

The 3A' reporter system was used to analyze the upstream and downstream context effects on translations termination. (A) Sequences that were predicted to provide a strong or weak context were placed upstream (5') and downstream (3') of the stop codon (in this case, UGA). The termination signals were placed between the second and third A' domains in the 3A' reporter plasmid. Expression from this plasmid generates two potential products, a termination product (14 kDa) and a readthrough product (21 kDa), which can be separated on the basis of size. (B) Expression of the 3A' reporter plasmid containing UGA stop signals in wild-type (white) and suppressor (black) tRNA strains is presented as percent readthrough. Combinations of strong (S) and weak (W) upstream and downstream contexts are illustrated (for example, a strong upstream and weak downstream signal is S-UGA-W). The error bars indicate standard errors of the mean.