Abstract
The intron-containing tRNA(Trp) precursor from Halobacterium volcanii, like many intron-containing archaebacterial precursor tRNAs, can assume a structure in which the two intron endonuclease cleavage sites are localized in two three-nucleotide loops separated by four base pairs. To investigate the role of this structure in cleavage by the halophilic endonuclease, a series of mutant tRNA(Trp) RNAs were prepared and evaluated as substrates. We find that alterations in this structure result in the loss of cleavage at both 5' and 3' sites. Cleavage of a 35-nucleotide model RNA substrate, containing only these features, demonstrates that sequences and structures present at the exon-intron boundaries are sufficient for recognition and cleavage. We have also examined the mechanism used by the halophilic endonuclease to identify the cleavage sites. Addition of a single base, or a base pair in the anticodon stem above the cleavage sites, does not affect the cleavage site selection. The addition of nucleotides between the two cleavage sites significantly decreases cleavage efficiency and has an effect on the cleavage site selection. These results demonstrate that the halophilic endonuclease requires a defined structure at the exon-intron boundaries and does not identify its cleavage sites by a measurement mechanism like that employed by eukaryotic tRNA intron endonucleases.
Highlights
The intron-containing tRNATrp precursor from Halobacterium volcanii, like many intron-containing archaebacterial precursor tRNAs, can assume a structure in which the two intron endonuclease cleavage sites are localized in two three-nucleotide loops separated by four base pairs
The addition of nucleotides between the two cleavage sites significantly decreases cleavage efficiency and has an effect on the cleavage site selection. These results demonstrate that the halophilic endonuclease requires a defined structure at the exon-intron boundaries and does not identify its cleavage sites by a measurement mechanism like that employed by eukaryotic tRNA intron endonucleases
We have previously shown that the H. volcanii tRNA endonuclease does not require the presence of mature tertiary structure in the intron-containing precursor or a complete intron; only those sequences and structures present at the exon-intron boundaries are required for recognition and cleavage [25]
Summary
The intron-containing tRNATrp precursor from Halobacterium volcanii, like many intron-containing archaebacterial precursor tRNAs, can assume a structure in which the two intron endonuclease cleavage sites are localized in two three-nucleotide loops separated by four base pairs. These results demonstrate that the halophilic endonuclease requires a defined structure at the exon-intron boundaries and does not identify its cleavage sites by a measurement mechanism like that employed by eukaryotic tRNA intron endonucleases. Comparative sequence analysis of archaebacterial intron-containing pretRNAs revealed that the cleavage sites were often located in two, three-nucleotide bulge loops, separated by four base pairs suggesting that this structure could function as a recognition element [26].
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