ABSTRACT
Tetracycline resistance protein Tet(O), which protects the bacterial ribosome from binding the antibiotic tetracycline, is a translational GTPase with significant similarity in both sequence and structure to the elongation factor EF-G. Here, we present an atomic model of the Tet(O)-bound 70S ribosome based on our cryo-electron microscopic reconstruction at 9.6-Å resolution. This atomic model allowed us to identify the Tet(O)-ribosome binding sites, which involve three characteristic loops in domain 4 of Tet(O). Replacements of the three amino-acid tips of these loops by a single glycine residue result in loss of Tet(O)-mediated tetracycline resistance. On the basis of these findings, the mechanism of Tet(O)-mediated tetracycline resistance can be explained in molecular detail.
Subject(s)
Bacterial Proteins/metabolism , Campylobacter jejuni/metabolism , Carrier Proteins/metabolism , Ribosomal Proteins/metabolism , Tetracycline Resistance , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/ultrastructure , Carrier Proteins/chemistry , Carrier Proteins/ultrastructure , Cryoelectron Microscopy , Guanosine Triphosphate/analogs & derivatives , Guanosine Triphosphate/chemistry , Guanosine Triphosphate/metabolism , Models, Molecular , Molecular Sequence Data , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , RNA, Transfer/chemistry , RNA, Transfer/metabolism , Ribosomal Proteins/chemistry , Ribosome Subunits, Large, Bacterial/metabolism , Ribosome Subunits, Large, Bacterial/ultrastructure , Ribosome Subunits, Small, Bacterial/metabolism , Ribosome Subunits, Small, Bacterial/ultrastructure , Ribosomes/metabolism , Ribosomes/ultrastructure , Structural Homology, ProteinABSTRACT
BACKGROUND: Several cis-acting regulatory sequences functioning at the level of mRNA or nascent peptide and specifically influencing transcription or translation have been described. These regulatory elements often respond to specific chemicals. RESULTS: We have developed a method that allows us to select cis-acting regulatory sequences that respond to diverse chemicals. The method is based on the beta-lactamase gene containing a random sequence inserted into the beginning of the ORF. Several rounds of selection are used to isolate sequences that suppress beta-lactamase expression in response to the compound under study. We have isolated sequences that respond to erythromycin, troleandomycin, chloramphenicol, meta-toluate and homoserine lactone. By introducing synonymous and non-synonymous mutations we have shown that at least in the case of erythromycin the sequences act at the peptide level. We have also tested the cross-activities of the constructs and found that in most cases the sequences respond most strongly to the compound on which they were isolated. CONCLUSIONS: Several selected peptides showed ligand-specific changes in amino acid frequencies, but no consensus motif could be identified. This is consistent with previous observations on natural cis-acting peptides, showing that it is often impossible to demonstrate a consensus. Applying the currently developed method on a larger scale, by selecting and comparing an extended set of sequences, might allow the sequence rules underlying the activity of cis-acting regulatory peptides to be identified.
Subject(s)
Base Sequence , Regulatory Sequences, Nucleic Acid , 4-Butyrolactone/analogs & derivatives , 4-Butyrolactone/pharmacology , Amino Acid Motifs/genetics , Chloramphenicol/pharmacology , Erythromycin/pharmacology , Gene Expression/drug effects , Genes, Reporter , Molecular Sequence Data , Open Reading Frames , Peptide Library , Peptides/genetics , Protein Synthesis Inhibitors/pharmacology , RNA, Messenger/chemistry , RNA, Messenger/genetics , RNA, Messenger/metabolism , Troleandomycin/pharmacology , beta-Galactosidase/genetics , beta-Galactosidase/metabolismABSTRACT
BACKGROUND: RNase III is a dsRNA specific endoribonuclease which is involved in the primary processing of rRNA and several mRNA species in bacteria. Both primary structural elements and the secondary structure of the substrate RNA play a role in cleavage specificity. RESULTS: We have analyzed RNase III cleavage sites around both ends of pre-23 S rRNA in the ribosome and in the protein-free pre-rRNA. It was found that in the protein-free pre-23 S rRNA the main cleavage site is at position (-7) in respect of the mature 5' end. When pre-23 S rRNA was in 70 S ribosomes or in 50 S subunits, the RNase III cleavage occurred at position (-3). We have demonstrated that RNase III interacts with both ribosomal subunits and with even higher affinity with 70 S ribosomes. Association of RNase III with 70 S ribosomes cannot be dissociated by poly(U) RNA indicating that the binding is specific. CONCLUSIONS: In addition to the primary and secondary structural elements in RNA, protein binding to substrate RNA can be a determinant of the RNase III cleavage site.
