Specialized ribosomes allow for the study of mutations in functionally important regions in 16 S rRNA, without affecting cell growth. The identification of functional regions in the central domain of 16S rRNA.
In order to identify the sequences in the central domain of 16 S rRNA of Escherichia coli that are important for ribosome function, we have generated random mutations using a PCR-based mutagenesis technique. We show that the effects of such mutations on ribosomal activity can be analyzed in vivo utilizing the specialized ribosome system. With this system the effect of rRNA mutations on ribosomal activity can be studied by measuring the translation of a modified CAT-mRNA by specialized ribosomes. Specialized ribosomes do not translate the endogenous mRNAs and, therefore, are expected to constitute a non-essential pool of ribosomes within the cell. In total, we have isolated 28 different clones harboring specialized ribosomes with single or multiple point mutations. We demonstrate that for none of these clones was cell growth retarded, even though some of the mutations severely impaired the activity of the specialized ribosomes, to as low as 3% of the wild-type level. For all mutants, their individual activities ranged between 3% and 100% of that of the wild-type activity. Comparison of several mutants indicates that mutations within the hairpin loops 787-795 and 898-901 strongly reduce the ribosomal activity. We also present evidence that the single-stranded region centered around residue A815 may be involved in maintaining translational accuracy.
Brink
M F
MF
Department of Biochemistry, Gorlaeus Laboratories, Leiden University, The Netherlands.
Nels
R N
RN
Verbeet
M P
MP
de Boer
H A
HA
D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000378:metabolism; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000378:metabolism; D012336:RNA, Ribosomal, 16S / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*
D017931:DNA Primers; D012333:RNA, Messenger; D012336:RNA, Ribosomal, 16S; D015500:Chloramphenicol O-Acetyltransferase