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A S Mankin

Publications and source records attributed to A S Mankin.

At least 37 records · Page 2Linked to original sources
PubMed1998

The antibiotic thiostrepton inhibits a functional transition within protein L11 at the ribosomal GTPase centre.

A newly identified class of highly thiostrepton-resistant mutants of the archaeon Halobacterium halobium carry a missense mutation at codon 18 within the gene encoding ribosomal protein L11. In the mutant proteins, a proline, conserved in archaea and bacteria, is converted to either serine or threonine. The mutations do not impair either the assembly of the mutant L11 into 70 S ribosomes in vivo or the binding of thiostrepton to ribosomes in vitro. Moreover, the corresponding mutations at proline 22, in a fusion protein of L11 from Escherichia coli with glutathione-S-transferase, did not reduce the binding affinities of the mutated L11 fusion proteins for rRNA of of thiostrepton for the mutant L11-rRNA complexes at rRNA concentrations lower than those prevailing in vivo. Probing the structure of the fusion protein of wild-type L11, from E. coli, using a recently developed protein footprinting technique, demonstrated that a general tightening of the C-terminal domain occurred on rRNA binding, while thiostrepton produced a footprint centred on tyrosine 62 at the junction of the N and C-terminal domains of protein L11 complexed to rRNA. The intensity of this protein footprint was strongly reduced for the mutant L11-rRNA complexes. These results indicate that although, as shown earlier, thiostrepton binds primarily to 23 S rRNA, the drug probably inhibits peptide elongation by impeding a conformational change within protein L11 that is important for the function of the ribosomal GTPase centre. This putative inhibitory mechanism of thiostrepton is critically dependent on proline 18/22. Moreover, the absence of this proline from eukaryotic protein L11 sequences would account for the high thiostrepton resistance of eukaryotic ribosomes.

Amino Acid Sequence↗
PubMed1997

Pactamycin resistance mutations in functional sites of 16 S rRNA.

Mutants of an archaeon Halobacterium halobium, resistant to the universal inhibitor of translation, pactamycin, were isolated. Pactamycin resistance correlated with the presence of mutations in the 16 S rRNA gene of H. halobium single rRNA operon. Three types of mutations were found in pactamycin resistant cells, A694G, C795U and C796U (Escherichia coli 16 S rRNA numeration) located distantly in rRNA primary structure but probably neighboring each other in the three-dimensional structure. Pactamycin resistance mutations either overlapped (C795U) or were located in the immediate vicinity of nucleotides protected by the drug in E. coli and H. halobium 16 S rRNA indicating that corresponding rRNA sites might be directly involved in pactamycin binding. Ribosomal functions were not affected significantly either by mutation of C795 (one of the positions protected by the P-site-bound tRNA), or by mutations of A694 and C796 (which neighbor nucleotides protected by tRNA) suggesting that tRNA-dependent protections of C795 and G693 are explained by a conformational change in the ribosome induced by the P-site-bound tRNA. A novel mode of pactamycin action is proposed suggesting that pactamycin restricts structural transitions in 16 S rRNA preventing the ribosome from adopting a functional conformation induced by tRNA binding.

Base Sequence↗
PubMed1997

Erythromycin resistance peptides selected from random peptide libraries.

Translation of a 5-codon mini-gene encoded in Escherichia coli 23 S rRNA was previously shown to render cells resistant to erythromycin (Tenson, T., DeBlasio, A., and Mankin, A. S. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5641-5646). Erythromycin resistance was mediated by a specific interaction of the 23 S rRNA-encoded pentapeptide with the ribosome. In the present study, peptides conferring erythromycin resistance were selected from in vivo expressed random peptide libraries to study structural features important for peptide activity. Screening of a 21-codon mini-gene library (the general structure ATG (NNN)20 TAA) demonstrated that only short peptides (3-6 amino acids long) conferred erythromycin resistance. Sequence comparison of erythromycin resistance peptides isolated from the 5-codon library (ATG (NNN)4 TAA) revealed a strong preference for leucine or isoleucine as a third amino acid and a hydrophobic amino acid at the C terminus of the peptide. When tested against other antibiotics, erythromycin resistance peptides rendered cells resistant to other macrolides, oleandomycin and spiramycin, but not to chloramphenicol or clindamycin. Defining the consensus amino acid sequence of erythromycin resistance peptides provided insights into a possible mode of peptide action and the nature of the peptide binding site on the ribosome.

Chromosome Mapping↗
PubMed1996

Mutations in the peptidyl transferase center of 23 S rRNA reveal the site of action of sparsomycin, a universal inhibitor of translation.

Sparsomycin is a universal and powerful inhibitor of peptide bond formation which, in contrast to many other ribosome-targeted antibiotics, does not produce footprints on rRNA. A mutant of an archaeon Halobacterium halobium has been isolated that exhibits resistance to sparsomycin. Resistant cells possessed a mutation in the 23 S rRNA, where C2518 (C2499 in Escherichia coli) was substituted by U. Introduction of the C2518U mutation into the chromosomal 23 S rRNA gene of wild-type H. halobium rendered cells resistant to sparsomycin, demonstrating that a single nucleotide alteration in the rRNA is sufficient to confer resistance. Accordingly, ribosomes containing mutant 23 S rRNA exhibited increased tolerance to sparsomycin in vitro. Mutations of two other nucleotide positions in the peptidyl transferase center, C2471 and U2519 (C2452 and U2500 in E. coli), conferred resistance to low concentrations of sparsomycin. The location of the sparsomycin resistance mutations reveals the possible site of drug binding and/or action. Our findings provide further support for the idea that rRNA may be directly involved in interaction with antibiotics and the catalysis of the peptide bond formation.

Base Sequence↗
PubMed1996

Mutations in domain II of 23 S rRNA facilitate translation of a 23 S rRNA-encoded pentapeptide conferring erythromycin resistance.

Mutations in domain II of Escherichia coli 23 S rRNA that cause resistance to erythromycin do so in a manner fundamentally different from mutations at the drug binding site in domain V of the 23 S rRNA. The domain II mutations are located in a hairpin structure between nucleotides 1198 and 1247. This is close to a short open reading frame in the 23 S rRNA that encodes a pentapeptide (E-peptide) whose expression in vivo renders cells resistant to erythromycin. Therefore, a possible mechanism of resistance caused by domain II mutations may be related to an increased expression of the E-peptide. To test this hypothesis, a range of point mutations was generated in domain II of 23 S rRNA in the vicinity of the E-peptide open reading frame. We find a correlation between erythromycin resistance of the mutant clones and increased accessibility of the ribosome binding site of the E-peptide gene. Furthermore, the erythromycin resistance determinant in the mutants was shown to be confined to a small 23 S rRNA segment containing the coding region and the ribosome binding site of the E-peptide open reading frame. It thus appears that the domain II mutations mediate erythromycin resistance by increasing expression of the 23 S rRNA-encoded E-peptide.

Base Sequence↗
PubMed1994

Cross-hypersensitivity effects of mutations in 23 S rRNA yield insight into aminoacyl-tRNA binding.

Halobacterium halobium cells contain one set of rRNA genes per genome. They have been used to characterize spontaneous mutants, carrying single nucleotide mutations in their rRNAs, that are resistant to different ribosomal antibiotics. Here we demonstrate that two different mutants that show resistance to thiostrepton, an inhibitor of the ribosomal GTPase-centre, are hypersensitive to amicetin, and other antibiotics, which act at the peptidyl transferase centre. Conversely, an amicetin-resistant mutant exhibits hypersensitivity to thiostrepton. A model is presented in which the two mutated sites, which are widely separated in the primary and secondary structure of 23 S rRNA, both participate in A-site binding of aminoacyl-tRNA.

Anti-Bacterial Agents↗
PubMed1994

A conserved secondary structural motif in 23S rRNA defines the site of interaction of amicetin, a universal inhibitor of peptide bond formation.

The binding site and probable site of action have been determined for the universal antibiotic amicetin which inhibits peptide bond formation. Evidence from in vivo mutants, site-directed mutations and chemical footprinting all implicate a highly conserved motif in the secondary structure of the 23S-like rRNA close to the central circle of domain V. We infer that this motif lies at, or close to, the catalytic site in the peptidyl transfer centre. The binding site of amicetin is the first of a group of functionally related hexose-cytosine inhibitors to be localized on the ribosome.

Anti-Bacterial Agents↗
PubMed1993

Heterogeneity of small plasmids from halophilic archaea.

Small multicopy plasmids in three strains of halophilic archaea, SB3, GRB, and GN101, were found to be present in a cell as a population of related but not identical sequences. Two types of heterogeneity were observed: macroheterogeneity, represented by two major plasmid sequence versions homologous to each other by 80%, and microheterogeneity, in which individual plasmids differ by one or a few nucleotide substitutions.

Amino Acid Sequence↗
PubMed1992

Introducing mutations into the single-copy chromosomal 23S rRNA gene of the archaeon Halobacterium halobium by using an rRNA operon-based transformation system.

A vector-transformation system is described that permits replacement of a portion of the single rRNA operon of the archaeon Halobacterium halobium with a homologous fragment from a vector-borne gene. The vector construct contains three functional sections: (i) an entire H. halobium rRNA operon with two selective mutations in the 23S rRNA gene, the substitutions of A----G at position 1159 conferring resistance to thiostrepton and C----U at position 2471 conferring resistance to anisomycin; (ii) the complete pHSB1 plasmid from Halobacterium sp. SB3, which interferes with vector maintenance in the transformed halobacterial cells; and (iii) a segment of the pBR322 plasmid that permits vector replication in Escherichia coli. Transformation of H. halobium with the vector plasmid generates cells resistant to both anisomycin and thiostrepton that can be selected for, and discriminated from spontaneous mutants, by a two-step selection procedure. After transformation, the plasmid recombines homologously with the chromosome so that the plasmid-borne rDNA segment with resistance markers substitutes for the corresponding region of the chromosomal rRNA operon, and the transforming plasmid is lost. Eventually, this leads to a homogeneous population of the mutant ribosomes in the cell. Other mutations that are engineered in the vector-borne rRNA sequences can be transferred to the chromosomal rRNA operon concomitantly with the selective markers. The system has considerable potential for ribosomal engineering.

Anisomycin↗
PubMed1991

Archaeal rRNA operons.

Ribosomal RNA (rRNA) operons of the archaea reflect both the unity and the diversity of this third primary taxon. They have proven to be a rich source of both molecular biological and phylogenetic information.

Archaea↗
PubMed1991

Chloramphenicol resistance mutations in the single 23S rRNA gene of the archaeon Halobacterium halobium.

A broad range of antibiotics affecting protein biosynthesis were screened for their ability to inhibit growth of the archaeon Halobacterium halobium. Only a few drugs, including chloramphenicol, produced inhibitory effects. Mutants which showed increased resistance to chloramphenicol were isolated; of the nine tested, eight exhibited a C----U transition at position 2471 and the ninth had an A----C transversion at position 2088 of 23S rRNA. A double mutant containing both C----U (position 2471) and A----C (position 2088) mutations was isolated, but the level of its chloramphenicol resistance did not exceed that of either single-point mutant. Inferences are made concerning the functional significance of the conserved nucleotides in rRNAs.

Base Sequence↗
PubMed1989

Ribosomal protein gene cluster of Halobacterium halobium: nucleotide sequence of the genes coding for S3 and L29 equivalent ribosomal proteins.

A 1643 base pair fragment encoding the S3 and L29 equivalent ribosomal proteins has been sequenced from the archaebacterium Halobacterium halobium. The incomplete open reading frame present upstream from the S3 gene encodes a protein homologous to the eubacterial ribosomal protein L22. The initiation codons of the S3 and L29 genes overlap with the termination codons of the upstream genes. A tight physical organization suggests that these genes are transcribed as a polycistronic operon. Peculiarities of the protein structure and gene organization are discussed.

Amino Acid Sequence↗
PubMed1989

Unusual nucleotide sequence heterogeneity of small multicopy pHSB plasmid from Halobacterium strain SB3, an archaebacterium.

A small multicopy plasmid, pHSB, from the Halobacterium strain SB3 is present in the cell as a population of molecules differing from each other by one or several point nucleotide substitutions. At least three sequence versions of the plasmid coexist in the cell. The pHSB primary structure heterogeneity may be a reflection of high genetic instability of the host organism.

Archaea↗
PubMed1988

Complex promoter pattern of the single ribosomal RNA operon of an archaebacterium Halobacterium halobium.

The transcription of the single ribosomal RNA gene cluster is initiated at an assemblage of promoters five of which were previously mapped in the 910 base pair region in front of the 16S rRNA gene. The nucleotide sequence between positions -1680 and -910 was determined and S1 nuclease mapping revealed therein two sites corresponding to the 5' ends of the primary transcripts. These two presumably represent the most distal promoters, P1 and P2, of the operon, P1 being separated from the 16S rRNA gene by more than 1250 bp. An additional putative "internal" promoter located within the operon body in the 16S/23S intergenic spacer was revealed by S1 nuclease mapping. A 528 bp long open reading frame is co-transcribed with the rRNA genes from the P1 promoter. Its possible functional implication is discussed.

Base Sequence↗