A conformational switch involving the 915 region of Escherichia coli 16 S ribosomal RNA (original) (raw)
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Journal of Molecular Biology, 1997
We describe the locations of sites within the 3D model for the 16 S rRNA (described in two accompanying papers) that are implicated in ribosomal function. The relevant experimental data originate from many laboratories and include sites of foot-printing, cross-linking or mutagenesis for various functional ligands. A number of the sites were themselves used as constraints in building the 16 S model. (1) The foot-print sites for A site tRNA are all clustered around the anticodon stem ± loop of the tRNA; there is no``allosteric'' site.
Rotational and Conformational Dynamics of Escherichia coli Ribosomal Protein L7/L12
Biochemistry, 1996
Fluorescence methods were utilized to study dynamic aspects of the 24 kDa dimeric Escherichia coli ribosomal protein L7/L12. Oligonucleotide site-directed mutagenesis was used to introduce cysteine residues at specific locations along the peptide chain, in both the C-terminal and N-terminal domains, and various sulfhydryl reactive fluorescence probes ((iodoacetamido)fluorescein, IAEDANS, pyrenemethyl iodoacetate) were attached to these residues. In addition to the full-length proteins, a hinge-deleted variant and variants corresponding to the C-terminal fragment and the N-terminal fragment were also studied. Both steady-state and time-resolved fluorescence measurements were carried out, and the results demonstrated that L7/L12 is not a rigid molecule. Specifically, the two C-terminal domains move freely with respect to one another and with respect to the dimeric N-terminal domain. Removal of the hinge region, however, significantly reduces the mobility of the C-terminal domains. The data also show that the rotational relaxation time monitored by the fluorescent probe depends upon the probe's excited state lifetime. This observation is interpreted to indicate that a hierarchy of motions exists in the L7/L12 molecule including facile motions of the C-terminal domains and dimeric N-terminal domain, in addition to the overall tumbling of the protein. Probes attached to the N-terminal domain exhibit global rotational relaxation times consistent with the molecular mass of the dimeric N-terminal fragment. Upon reconstitution of labeled L7/L12 with ribosomal cores, however, the motion associated with the dimeric N-terminal domain is greatly diminished while the facile motion of the C-terminal domains is almost unchanged.
Journal of Molecular Biology - J MOL BIOL, 1997
We describe the locations of sites within the D model for the 16 S rRNA (described in two accompanying papers) that are implicated in ribosomal function. The relevant experimental data originate from many laboratories and include sites of foot-printing, cross-linking or mutagenesis for various functional ligands. A number of the sites were themselves used as constraints in building the 16 S model. (1) The foot-print sites for A site tRNA are all clustered around the anticodon stem-loop of the tRNA; there is no “allosteric” site. (2) The foot-print sites for P site tRNA that are essential for P site binding are similarly clustered around the P site anticodon stem-loop. The foot-print sites in 16 S rRNA helices 23 and 24 are, however, remote from the P site tRNA. (3) Cross-link sites from specific nucleotides within the anticodon loops of A or P site-bound tRNA are mostly in agreement with the model, whereas those from nucleotides in the elbow region of the tRNA (which also exhibit ex...
Molecular Cell, 2003
Noller et al., 1992; Garrett and Rodriguez-Fon-Weizmann Institute seca, 1995; Samaha et al., 1995). Crystal structures of 76100 Rehovot complexes of Thermus thermophilus ribosomes (T70S) Israel with tRNA (Yusupov et al., 2001) as well as of the large 2 Max-Planck-Research Unit for Ribosomal Structure ribosomal subunits from the archaeon Haloarcula maris-Notkestrasse 85 mortui (H50S) and the mesophilic eubacterium Deino-22603 Hamburg coccus radiodurans (D50S) with various substrate pepti-Germany dyl-transferase analogs or inhibitors (Nissen et al., 2000; 3 Max-Planck-Institute for Molecular Genetics Schluenzen et al., 2001; Schmeing et al., 2002; Hansen Ihnestrasse 73 et al., 2002) show that the PTC can be described as a 4 FB Biologie, Chemie, Pharmazie pocket with a tunnel emerging from it. It is located at Frei University Berlin the bottom of a cavity containing all of the nucleotides Takustrasse 3
A deletion mutation at the 5′ end of Escherichia coli 16S ribosomal RNA
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1990
A deletion of five nucleotides was introduced at the 5' end of the Escherichia coli 16S rRNA gene cloned in an appropriate vector under control of a T7 promoter. The 16S rRNA generated by in vitro transcription could be assembled into 30S subunits. The deletion did not affect the efficiency of translation of natural messengers and the correct selection of the reading frame. However, it reduced the binding of the messengers, which suggests that the 5' end of 16S rRNA is located on the pathway followed by the messengers on the 30S subunits. The deletion also restricted the stimulation of misreading by streptomycin in a poly(U)-directed system. This is in accord with the proximity of the 5' end of 168 rRNA to proteins 84, 5andS12,whichareknowntobeinvolvedinthecontroloftranslationalaccuracy.0167−4781/90/5 and S12, which are known to be involved in the control of translational accuracy. 0167-4781/90/5andS12,whichareknowntobeinvolvedinthecontroloftranslationalaccuracy.0167−4781/90/03.50
A mutation in the 530 loop ofEscherichia coli16S ribosomal RNA causes resistance to streptomycin
Nucleic Acids Research, 1988
Oligonucleotide-directed mutagenesis was used to introduce an A to C transversion at position 523 in the 16S ribosomal RNA gene of Escherichia coli rrnB operon cloned in plasmid pKK3535. E. coli cells transformed with the mutated plasmid were resistant to streptomycin. The mutated ribosomes isolated from these cells were not stimulated by streptomycin to misread the message in a poly(U)-directed assay. They were also restrictive to the stimulation of misreading by other error-promoting related aminoglycoside antibiotics such as neomycin, kanamycin or gentamicin, which do not compete for the streptomycin binding site. The 530 loop where the mutation in the 16S rRNA is located has been mapped at the external surface of the 30S subunit, and is therefore distal from the streptomycin binding site at the subunit interface. Our results support the conclusion that the mutation at position 523 in the 16S rRNA does not interfere with the binding of streptomycin, but prevents the drug from inducing conformational changes in the 530 loop which account for its miscoding effect. Since this effect primarily results from a perturbation of the translational proofreading control, our results also provide evidence that the 530 loop of the 16S rRNA is involved in this accuracy control.