
Full text loading...
Category: History of Science; Microbial Genetics and Molecular Biology
Adventures with Frameshift Suppressor tRNAs, Page 1 of 2
< Previous page | Next page > /docserver/preview/fulltext/10.1128/9781555816810/9781555815387_Chap14-1.gif /docserver/preview/fulltext/10.1128/9781555816810/9781555815387_Chap14-2.gifAbstract:
During the 1960s it was established that the modified nucleosides present in tRNA and rRNA are synthesized after the primary transcript is made. To obtain tRNA lacking only one modified nucleoside, a mutant defective in the synthesis of one modified nucleoside is required. This paper inspired the author to use a transposon to obtain mutants defective in tRNA methylation. Using such a method, the author discovered the metabolic link between the synthesis of aromatic amino acids and the synthesis of uridine-5-oxyacetic acid (cmo5U) and its methyl ester (mcmo5U). Provided that the tRNA(mcmo5U34)methyltransferase is a multimeric complex, poor translation termination mediated by a defective RF2 would extend the CmoA peptide, making it unable to form a potential complex required for the tRNA(mcmo5U34)methyltransferase activity. The sufC10 as well as sufC13 and sufC14 mutants have suppressor specificity similar to that induced by sufA6 and sufB2. It later emerged that these mutants each contain mutations in two genes: sufX and sufY. The sufC10 mutants contain the sufX201 and sufY204 mutations; sufC13 mutants, the sufX202 and sufY205 mutations; and the sufC14 mutant, the sufX203 and sufY206 mutations. The frameshift tRNA suppressors isolated more than 30 years ago in the laboratory of John Roth have clearly been important tools to study how the ribosome maintains the reading frame. Recent analyses using this collection of frameshift suppressor mutants have revealed new facts of the operational mechanism behind this important and conserved feature of translation.
Full text loading...
Synthesis of cmo5U34 and its methyl ester mcmo5U34 and the link to the synthesis of aromatic amino acids and vitamins ( 20 ). Gray arrows indicate the link between chorismic acid (or an unknown derivative of it) and different steps in the synthesis of cmo5U. CmoA possesses an AdoMet binding site. It is likely to be an AdoMet-dependent methyltransferase. Since only one of the carbon atoms in cmo5U originates from Met ( 11 ), the CmoA polypeptide may be part of a complex also mediating the formation of the methylester (mcmo5U) of cmo5U.
(A) The genetic code. The shaded codon boxes are the family codon boxes, which contain four codons representing one amino acid. The six family codon boxes in the lighter shade contain tRNAs having cmo5U or mcmo5U as the wobble nucleoside. The codon boxes with a white background are the mixed codon boxes. (B) The proline family codon box (CCN). proK, proL, and proM denote the genes encoding , , and , respectively, and the wobble nucleoside, which is present in position 34, is indicated. The circles correspond to the codon read by a tRNA, and a line connecting two or more circles indicates that the same tRNA reads those codons (e.g., the proL contains G34 as the wobble nucleoside and reads the CCC and CCU codons). The black circles show the codon reading abilities predicted by the wobble hypothesis ( 9 ) and the revised wobble rules ( 40 ). The gray circle for proM (codon CCC) indicates that this tRNA reads CCC codons provided that cmo5U is present ( 20 ). The proM , having ho5U instead of cmo5U as the wobble nucleoside, reads this codon and also CCU less efficiently than the fully modified tRNA as judged by adequate growth rate comparisons ( 20 ). Even in the presence of proL and proK , ho5U instead of cmo5U in the proM reduces the A-site selection at the CCC codon but not at the CCU codon ( 21 ). Thus, in cells having a normal Pro-tRNA population, the presence of cmo5U34 is important for decoding CCC. This is most likely also true for a tRNA having U as the wobble nucleoside, which some of the tRNAs might have in an aro mutant ( 11 ). (Adapted from reference 20 .)
Three models showing how a defective tRNA induces frameshifting in the P-site. (A) The defective tRNA (indicated by a gray diamond) is too slow (indicated by a broken line) in entering the A-site, allowing a third position mismatched tRNA (depicted by a black bar at the wobble position) to decode the A-site codon. After a normal three-nucleotide translocation to the P-site, the third position mismatched tRNA is prone to slip into an overlapping reading frame. (B) The defective tRNA (indicated by a gray diamond) decodes the codon in the A-site, but once it has been translocated into the P-site it may slip on the mRNA. (C) The defective tRNA (indicated by a gray diamond) is too slow (indicated by a broken line) in entering the A-site, providing a pause that allows the cognate P-site tRNA to slip. Broken arrows indicate a slow entry into the +1 frame compared to continued reading in the zero frame. The original (zero) reading frame is indicated in the mRNA with alternating black and gray triplets. “Defective” can either indicate alterations in the primary sequence or hypo-modification of the tRNA. (Reprinted from reference 22 with permission from Elsevier.)