Use of tRNA suppressors to probe regulation of Escherichia coli release factor 2

Recognition of the mRNA selenocysteine insertion sequence by the specialized translational elongation factor SELB.

“…suppress nonsense codons have been described (Engelberg 1981;Dahlberg 1989;Brown et al 1990;Rein and Levin 1992). For instance, the suppressible, in-frame UGA codon in the E. coli RF2 message is dependent on an adjacent Shine-Dalgarno-like element (Craigen et al 1985;Curran and Yarus 1988), whereas the m a m m a l i a n type C retroviruses also rely on a cis-acting m R N A elem e n t for recoding an in-frame U G A codon from nonsense to glutamate during synthesis of the gag-pol gene product (Rein and Levin 1992). Like selenocysteine, the type C retroviruses use a unique t R N A (Rein and Levin 1992) although specialized translation factors have not been identified.…”

Suppression of eukaryotic translation termination by selected RNAs

“…Sense triplets are decoded on the ribosome by specific transfer RNAs via base pairing between codons in mRNA and anticodon triplets in tRNA+ In contrast, nonsense (termination, stop) codons are decoded in two canonical ways+ UGA can be decoded as selenocysteine by a specialized tRNA-translation factor complex (selenocysteinyl-tRNA Sec : SelB) that directs SectRNA Sec to a unique message structure (Hüttenhofer & Bock, 1998)+ More frequently UGA, UAA, and UAG are decoded as stop-translation signals+ A single-polypeptide release factor (RF) termed eRF1 performs this function in eukaryotes and two proteins, RF1 and RF2, do so in bacteria (reviewed in Tate et al+, 1996;Buckingham et al+, 1997;)+ The class-1 release factor proteins RF1, RF2, and eRF1, are distinguished by their ability to trigger peptidyl-tRNA hydrolysis, catalyzed by the ribosomal peptidyl transferase when a stop codon enters the A site+ Although the nature of this hydrolytic signal remains unknown, it is clear that class-1 RF proteins strongly compete with suppressor tRNAs in vitro and in vivo (Weiss et al+, 1984;Curran & Yarus, 1988;Eggertsson & Soll, 1988;Drugeon et al+, 1997;Le Goff et al+, 1997)+ They therefore probably overlap the ribosomal decoding site+ As has been suggested (Cantor, 1979;Nissen et al+, 1995;Ito et al+, 1996;Nakamura et al+, 1996;Kisselev et al+, 2000) and proven recently by crystallography (Song et al+, 2000), eRF1 adopts an extended, perhaps tRNA-like overall shape+ Other proteins acting at the ribosomal A site, such as elongation factor EF-G and bacterial ribosome recycling factor (RRF; Nyborg et al+, 1996;Selmer et al+, 1999), also emulate the tRNA shape+ These structural findings support a "tRNA-analog" hypothesis ; that is, that termination codons may be decoded directly by class-1 RFs, whose similar shape allows mimicry of tRNA action+ Thus RFs may contact stop codons in the decoding (A) site on the ribosome+ These facts suggest that RF action would be effectively inhibited by other strongly bound RNAs+ Selected RNA ligands for RF protein(s) therefore might act as highly specific inhibitors of stop codon translation+ This would be useful for biochemical dissection of the pathway for translation termination+ In addition, be-cause a single eRF1 protein decodes all termination codons, such RNA ligands would likely comprise a new type of omnipotent termination suppressor, increasing stop codon readthrough by interfering with the usual termination pathway+ Therefore we have isolated eRF ligands using selection-amplification (for review, see Wilson & Szostak, 1999)+ Starting with a randomized mixture of oligoribonucleotides, repetitive selection for RNA-RF binding yielded RNAs with high affinity ...…”

The second to last amino acid in the nascent peptide as a codon context determinant.

“…The prJB mRNA, which codes for release factor 2 (RF-2) in Escherichia coli, has an in-frame UGA codon in its coding region which is bypassed by a frameshift event giving the mature gene product (Craigen and Caskey, 1986;Donly et al, 1990). One feature of this autoregulatory mechanism is an interaction between the anti-ShineDalgarno region of the 16S rRNA and a six base Shine-Dalgarno element two codons upstream of the UGA codon (Weiss et al, 1987(Weiss et al, , 1988Curran and Yarus, 1988).…”