TY - JOUR
T1 - Allele-specific structure probing of plasmid-derived 16S ribosomal RNA from Escherichia coli
AU - Powers, Ted
AU - Noller, Harry F.
PY - 1993/1/15
Y1 - 1993/1/15
N2 - Biochemical analysis of Escherichia coli ribosomes containing mutant 16S or 23S (r)ribosomal RNAs, produced via cloned rDNA genes on multicopy plasmids, has been hindered by the background of wild-type (wt) ribosomes containing host-derived rRNA. Here, we describe a method for the construction of unique priming sites in 16S rRNA that allow allele-specific structure probing of ribosomes containing plasmid-encoded RNA. Phenotypically silent mutations, designed to mimic related eubacterial sequences, have been introduced into four phylogenetically variable regions in the 16S rDNA gene that allow inspection of several 16S rRNA functional sites. When oligodeoxyribonucleotides complementary to these altered sequences are used to prime cDNA synthesis in primer extension reactions using reverse transcriptase, only plasmid-derived 16S rRNA is used as a template, thus rendering the wt background invisible. Unexpectedly, we were unable to introduce silent mutations into one nonconserved helix in 16S rRNA, suggesting that constraints in addition to Watson-Crick pairing are important in this region.
AB - Biochemical analysis of Escherichia coli ribosomes containing mutant 16S or 23S (r)ribosomal RNAs, produced via cloned rDNA genes on multicopy plasmids, has been hindered by the background of wild-type (wt) ribosomes containing host-derived rRNA. Here, we describe a method for the construction of unique priming sites in 16S rRNA that allow allele-specific structure probing of ribosomes containing plasmid-encoded RNA. Phenotypically silent mutations, designed to mimic related eubacterial sequences, have been introduced into four phylogenetically variable regions in the 16S rDNA gene that allow inspection of several 16S rRNA functional sites. When oligodeoxyribonucleotides complementary to these altered sequences are used to prime cDNA synthesis in primer extension reactions using reverse transcriptase, only plasmid-derived 16S rRNA is used as a template, thus rendering the wt background invisible. Unexpectedly, we were unable to introduce silent mutations into one nonconserved helix in 16S rRNA, suggesting that constraints in addition to Watson-Crick pairing are important in this region.
KW - chemical modification
KW - primer extension
KW - Ribosomes
KW - rRNA functional sites
KW - rRNA mutations
KW - site-directed mutagenesis
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U2 - 10.1016/0378-1119(93)90542-B
DO - 10.1016/0378-1119(93)90542-B
M3 - Article
C2 - 7678570
AN - SCOPUS:0027434504
VL - 123
SP - 75
EP - 80
JO - Gene
JF - Gene
SN - 0378-1119
IS - 1
ER -