TY - JOUR
T1 - Selection and characterization of randomly produced mutants in the gene coding for M1 RNA
AU - Lumelsky, Nadya
AU - Altman, Sidney
N1 - Funding Information:
We are grateful to D. Wesolowski for excellent technical assistance and to all members of our laboratory for helpful discussions. We thank R. Myers of the University of California at San Francisco for gifts of bacterial strains and phage, and for advice on chemical mutagenesis and selection of mutants. This work was supported by a U.S. NIH postdoctoral grant (to N.L.) and by grants from the U.S. NIH and U.S. NSF (to S.A.).
PY - 1988/8/5
Y1 - 1988/8/5
N2 - The gene for M1 RNA, the catalytic subunit of RNase P of Escherichia coli, was subjected to random chemical mutagenesis in vitro. Mutations were selected by electrophoresis in denaturing gradient gels. Twenty-seven different mutants of the gene for M1 RNA were selected, and in 24 cases the mutations were identified as single base substitutions. The mutant forms of M1 RNA were analyzed in vitro for catalytic activity in the absence and in the presence of the protein subunit of RNase P (C5 protein). The structure of mutant RNAs was probed by limited digestion with ribonuclease T1; a correlation between reduced catalytic activity of mutant M1 RNAs and perturbations in secondary and tertiary structure was noted in many cases. The results indicate the involvement of specific regions of the M1 RNA molecule in the catalytic function of RNase P, in the binding of the C5 protein, and in substrate binding.
AB - The gene for M1 RNA, the catalytic subunit of RNase P of Escherichia coli, was subjected to random chemical mutagenesis in vitro. Mutations were selected by electrophoresis in denaturing gradient gels. Twenty-seven different mutants of the gene for M1 RNA were selected, and in 24 cases the mutations were identified as single base substitutions. The mutant forms of M1 RNA were analyzed in vitro for catalytic activity in the absence and in the presence of the protein subunit of RNase P (C5 protein). The structure of mutant RNAs was probed by limited digestion with ribonuclease T1; a correlation between reduced catalytic activity of mutant M1 RNAs and perturbations in secondary and tertiary structure was noted in many cases. The results indicate the involvement of specific regions of the M1 RNA molecule in the catalytic function of RNase P, in the binding of the C5 protein, and in substrate binding.
UR - http://www.scopus.com/inward/record.url?scp=0023794579&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0023794579&partnerID=8YFLogxK
U2 - 10.1016/0022-2836(88)90277-X
DO - 10.1016/0022-2836(88)90277-X
M3 - Article
C2 - 2459394
AN - SCOPUS:0023794579
SN - 0022-2836
VL - 202
SP - 443
EP - 454
JO - Journal of molecular biology
JF - Journal of molecular biology
IS - 3
ER -