Two regions in the 3t domain of 16S rRNA (the RNA of the small ribosomal subunit) have been implicated in decoding of termination codons. One is located at nucleotide 1054 in helix 34 and the other is around nucleotide 1408 at the base of helix 44. Using segment-directed PCR random mutagenesis, I isolated 33 translational suppressor mutations in the 3t domain of 16S rRNA. All of the mutations were located in two helices, helix 34 and helix 44, out of the sixteen helices in the 3t domain of 16S rRNA, and they were clustered at five nucleotide positions of the total of 612 nucleotides in that domain. Four of the five mutant nucleotide positions are situated in either bulged or non-paired regions in the two helices, suggesting that these nucleotides are available for interactions with other molecules during protein synthesis. Characterization of the mutations by both genetic and biochemical methods indicated that some of the mutations are defective in UGA-specific peptide chain termination and that others may be defective in peptide chain termination at all termination codons. The studies of the mutations at an internal loop in the non-conserved region of helix 44 also indicated that this structure, in a non-conserved region of 16S rRNA, is involved in both peptide chain termination and assembly of 16S rRNA.
With a suppressible trpA UAG nonsense mutation, a spontaneously arising translational suppressor mutation was isolated in the rrnB operon cloned into a pBR322-derived plasmid. The mutation caused suppression of UAG at two codon positions in trpA but did not suppress UAA or UGA mutations at the same trpA positions. The specificity of the rRNA suppressor mutation suggests that it may cause a defect in UAG-specific peptide chain termination. The mutation is a single nucleotide deletion (G2484D) in helix 89 of 23S rRNA (the large RNA of the large ribosomal subunit). Helix 89, in domain V (of the six domains of 23S rRNA), is universally conserved and is one of the helices associated with the peptidyl-transferase center of the ribosome. Cells transformed with the UAG suppressor-containing plasmid can grow at 31¡ C and below but not at 35¡ C and above. Such a temperature-conditional lethal phenotype has been seen for other rRNA translational suppressors that exhibit codon specificity or preference. One such mutation is G1093A located in the GTPase center, in domain II of 23S rRNA. When the helix 89 mutation G2484D was combined with G1093A, in one cloned 23S rRNA gene, the first mutation reversed the UGA-specific suppression phenotype of the second, which has been shown in vitro to be defective in RF2 (UGA-specific)-dependent peptidyl-tRNA hydrolysis. The result indicates a functional interaction between two regions of 23S rRNA. Furthermore, it provides suggestive in vivo evidence for the involvement of the peptidyl-transferase center of 23S rRNA in peptide chain termination. The D2484 and A1093/D2484 (double) mutations were also observed to alter the decoding specificity of the suppressor tRNA lysT(U70), which has a mutation in its acceptor stem. That result suggests that there is an interaction between the stem-loop region of helix 89 of 23S rRNA and the acceptor stem of tRNA during decoding and that the interaction is important for the decoding specificity of tRNA.
Using gene manipulation procedures, I have constructed a new expression vector to express and purify the cellular protein factors required for a recently developed, realistic in vitro termination assay. The gene for each protein was cloned into the newly constructed vector in such a way that expression yielded a protein with an N-terminal affinity tag, for specific, rapid purification. The amino terminus was engineered so that, after purification, the unwanted N-terminal tag can be completely removed from the protein by thrombin cleavage, yielding a natural amino acid sequence for each protein. I have cloned all three release factor genes into this new expression vector and the genes for all the other protein factors into a pCAL-n expression vector. These constructs will allow our laboratory group to quickly and inexpensively purify all the protein factors needed for the new in vitro termination assay.
Functions and Intramolecular Interactions of Ribosomal RNA in Decoding of Termination Codons