Nucleic Acids Research, 1994, Vol. 22, No. 16 3347-3353
© 1994
ENZYMOLOGY |
The processing of wild type and mutant forms of rat nuclear pre-tRNALys by the homologous RNase P
Department of Biochemistry and Molecular Biophysics, Virginia Commonwealth University Richmond, VA 23298, USA
*To whom correspondence should be addressed
Received May 25, 1994. Revised July 20, 1994. Accepted July 20, 1994.
The 5' processing of rat pre-tRNALys and a series of mutant derivatives by rat cytosolic RNase P was examined. In standard, non-kinetic assays, mutant precursors synthesized in vitro with 5' leader sequences of 10, 17, 24, 25, and 46 nucleotides were processed to approximately equal levels and yielded precisely cleaved 5' processed intermediates with the normal 7-base pair aminoacyl stems. The construct containing the tRNALvs with the 46-nucleotide leader was modified by PCR to give a series of pre-tRNALvs mutants designed to measure the effect on processing by (1) substituting the nucleotide at the +1 position, (2) pairing and unpairing the +1 and + 72 bases, (3) elongating the aminoacyl stem, and (4) disrupting the helix of the aminoacyl stem. Comparative kinetic analyses revealed that changing the wild type 1G to A, C, or U was well tolerated by the RNase P provided that compensatory changes at + 72 created a base pair or a GU noncanonical pair. Mutants with elongated aminoacyl stems that were produced either by inserting an additional base pair at + 3:a + 69:a or by pairing the –1A with a + 73U, were processed to yield 7-base pair aminoacyl stems, but with different efficiencies. The efficiency seen with the double insertion mutant was higher than even the wild type precursor, but the – 1 A U + 73 mutant was a relatively poor substrate. Disrupting the aminoacyl stem helix by introducing a + 7G G + 66 mispairing or by inserting a single G at the + 3:a position dramatically reduced the processing efficiency, although the position of cleavage occurred precisely at the wild type cleavage site. However, the single insertion of a C at the +69:a position resulted in an efficiently cleaved precursor, but permitted a minor, secondary cleavage within the leader between the – 6 and – 5 nucleotides in addition to the dominant wild type scission.