On the mechanism of leftward frameshifting at several hungry codons.
We have used lacZ reporter genes to assess leftward ribosome frameshifting on sequences containing the quadruplet U UUC followed by several different triplets coding for lysine, isoleucine, or leucine. Limitation for lysine-tRNA provokes leftward frameshifting when the slippery quadruplet is followed by either lysine codon aag or aaa, but not when followed by an isoleucine or leucine codon. Limitation for isoleucine provokes frameshifting when the quadruplet is followed by either isoleucine codon aua or auc, but not when it is followed by a lysine codon. We conclude that the quadruplet promotes shifting when the ribosome is stalled at any "hungry" codon immediately after it. Changing the quadruplet to U AGC, at which peptidyl-tRNA cognate to the AGC triplet will be mismatched at all three anticodon positions if it slips left, abolishes frameshifting when the ribosome is stalled at the next position. We conclude that the U UUC quadruplet promotes frameshifting by virtue of its ability to pair with a left-slipped peptidyl-tRNA. The frameshift promoted by isoleucine-tRNA limitation of the U UUC aua sequence was analyzed by amino acid sequencing of the protein product. It occurs through reading of the Cau histidine codon overlapping the hungry codon from the left. This result rules out a "simultaneous slippage" type of mechanism. It strongly suggests instead that starvation-promoted frameshifting occurs primarily by slippage of peptidyl-tRNA just upstream of the stall site, followed by decoding of the triplet overlapping the stall site from the left or 5' side. A secondary finding is that the last base of the "hungry" codon has a moderate effect on its shiftiness, aag being shiftier than aaa, and aua being shiftier than auc.
Barak
Z
Z
Department of Life Sciences, Ben Gurion University of the Negev, Beersheva, Israel.
Lindsley
D
D
Gallant
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon / Q000737:chemistry / Q000235:genetics*; D018965:Frameshifting, Ribosomal / Q000235:genetics*; D005822:Genetic Vectors; D007532:Isoleucine / Q000031:analogs & derivatives / Q000494:pharmacology; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D007763:Lac Operon / Q000235:genetics; D008239:Lysine / Q000031:analogs & derivatives / Q000494:pharmacology; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D009843:Oligoribonucleotides / Q000737:chemistry; D012346:RNA, Transfer, Amino Acyl / Q000235:genetics / Q000378:metabolism; D012354:RNA, Transfer, His / Q000235:genetics / Q000378:metabolism; D012355:RNA, Transfer, Ile / Q000235:genetics / Q000378:metabolism; D012357:RNA, Transfer, Lys / Q000235:genetics / Q000378:metabolism; D017421:Sequence Analysis; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000378:metabolism
D003062:Codon; D009838:Oligodeoxyribonucleotides; D009843:Oligoribonucleotides; D012346:RNA, Transfer, Amino Acyl; D012354:RNA, Transfer, His; D012355:RNA, Transfer, Ile; D012357:RNA, Transfer, Lys; C019037:tRNA, peptidyl-; D007532:Isoleucine; C039763:lysine hydroxamate; C027007:isoleucylhydroxamic acid; D007544:Isopropyl Thiogalactoside; D001616:beta-Galactosidase; D008239:Lysine