Site-specific mutation of the conserved m6(2)A m6(2)A residues of E. coli 16S ribosomal RNA. Effects on ribosome function and activity of the ksgA methyltransferase.
In vitro synthesis of mutant 16S RNA and reconstitution with ribosomal proteins into a mutant 30S ribosome was used to make all possible single base changes at the universally conserved A1518 and A1519 residues. All of the mutant RNAs could be assembled into a ribosomal subunit which sedimented at 30 S and did not lack any of the ribosomal proteins. A series of in vitro tests of protein synthesis ability showed that all of the mutants had some activity. The amount varied according to the assay and mutant, but was never less than 30% and was generally above 50%. Therefore, neither the conserved A1518 nor A1519 residues are essential for ribosome function. The mutant ribosomes could also be methylated by the ksgA methyltransferase to 70-120% of the expected amount. Thus, neither of the A residues is required for methylation of the other, ruling out any obligate order of methylation of A1518 and A1519.
Cunningham
P R
PR
Roche Institute of Molecular Biology, Roche Research Center, Nutley, NJ 07110.
Weitzmann
C J
CJ
Nurse
K
K
Masurel
R
R
Van Knippenberg
P H
PH
Ofengand
J
J
D000227:Adenine Nucleotides / Q000235:genetics*; D001482:Base Composition; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D006860:Hydrogen Bonding; D007700:Kinetics; D008745:Methylation; D008780:Methyltransferases / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D015345:Oligonucleotide Probes; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D012270:Ribosomes / Q000378:metabolism*
D000227:Adenine Nucleotides; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D012336:RNA, Ribosomal, 16S; D008780:Methyltransferases; C047346:16S rRNA (adenine(1518)-N(6)-adenine(1519)-N(6))-dimethyltransferase