Splice junction mutations in a yeast tRNA gene which alter the rate and precision of processing.
We have introduced mutations into a tRNALeu3 gene which alter the intron boundaries and examined their effects on RNA splicing. Our results show that the 5'-proximal splice junction is not specified by the position of an adjacent base-paired stem present in all naturally occurring tRNA precursors. Also, efficient cleavage of 5'-splice junctions unique to these mutants, -CpU-, -UpA- and -UpG-, indicates the purine found at the 5'-side of this site in all natural precursors is dispensable. Some alterations of the sequence and structure at the 5'-proximal splice site reduce the rate of cleavage therein and result in accumulation of molecules composed of the 5'-half of the tRNA plus the intron. The precise position of the 5'-proximal cleavage site can vary +/- 1 base in these mutants. The 3'-proximal splice junction is rendered inactive by changing the prospective splice junction sequence from -ApC- to -CpC- and reducing the size of an unpaired loop at this site from six to two bases. Very small amounts of RNA composed of the 3'-half of the tRNA plus the intron accumulate from this precursor. We conclude that splice junction sequence and structure affect both the rate and precision of intervening sequence removal.
Chambers
J
J
Department of Molecular Biology, University of Wyoming, Laramie 82071.
Raymond
G J
GJ
Kim
D
D
Raymond
K C
KC
Nelson
C
C
Clark
S
S
Johnson
J D
JD
D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D004271:DNA, Fungal / Q000235:genetics; D005800:Genes, Fungal; D007438:Introns; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D009710:Nucleotide Mapping; D012322:RNA Precursors / Q000235:genetics; D012326:RNA Splicing; D012345:RNA, Transfer, Amino Acid-Specific / Q000235:genetics*; D012356:RNA, Transfer, Leu / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014158:Transcription, Genetic
D004271:DNA, Fungal; D012322:RNA Precursors; D012345:RNA, Transfer, Amino Acid-Specific; D012356:RNA, Transfer, Leu