TY - JOUR
T1 - Point mutations in the middle of 16S ribosomal RNA of E. coli produced by deletion loop mutagenesis
AU - Zwieb, Christian
AU - Dahlberg, Albert E.
N1 - Funding Information:
A3CNCWLEDGEMENTS We are grateful to Bruce Duncan for giving us the ung~ strain BD 817, to David Jemiolo, Melvin Santer and James Dahlberg for critical reading of the manuscript, and to G. Q. Pennabble for numerous discussions. This work was supported by a fellowship from the Deutsche Forschungsgemeinschaft to C.Z. and a USPHS grant GM 19756 from the National Institutes of Health to A.E.D.
PY - 1984/5/25
Y1 - 1984/5/25
N2 - Using the plasmid pKK3535, which contains the rrnB operon of Escherlchia coli in pBR322, a deletion mutation was constructed which lacks bases 822 to 874 in the middle of the 16S ribosomal RNA. This results in an "amputation" of a very distinct stem and loop structure in the RNA. By forming a heteroduplex between the deletion plasmid and the original pKK3535 and by modifying the single-stranded deletion loops with bisulfite, we produced plasmids containing one or two base changes at positions 839, 840, 841, 867 or 876. The clustering of the mutations near the top of the stem, and the inability to get base changes at other positions, suggests that single alterations at particular positions severely affect the formation of a functional ribosome. The ability to recover mutations at these positions is not determined by the secondary structure of the DNA during bisulfite mutagenesis. Restriction enzyme analysis of 12 revertants from a slow growing mutant (altered at positions 839 and 876) shows that they did not compensate for the mutation by re-establishing the original wild type sequence.
AB - Using the plasmid pKK3535, which contains the rrnB operon of Escherlchia coli in pBR322, a deletion mutation was constructed which lacks bases 822 to 874 in the middle of the 16S ribosomal RNA. This results in an "amputation" of a very distinct stem and loop structure in the RNA. By forming a heteroduplex between the deletion plasmid and the original pKK3535 and by modifying the single-stranded deletion loops with bisulfite, we produced plasmids containing one or two base changes at positions 839, 840, 841, 867 or 876. The clustering of the mutations near the top of the stem, and the inability to get base changes at other positions, suggests that single alterations at particular positions severely affect the formation of a functional ribosome. The ability to recover mutations at these positions is not determined by the secondary structure of the DNA during bisulfite mutagenesis. Restriction enzyme analysis of 12 revertants from a slow growing mutant (altered at positions 839 and 876) shows that they did not compensate for the mutation by re-establishing the original wild type sequence.
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U2 - 10.1093/nar/12.10.4361
DO - 10.1093/nar/12.10.4361
M3 - Article
C2 - 6328419
AN - SCOPUS:0021770595
VL - 12
SP - 4361
EP - 4375
JO - Nucleic Acids Research
JF - Nucleic Acids Research
SN - 0305-1048
IS - 10
ER -