Aminoglycoside antibiotics target the 16S ribosomal RNA (rRNA) bacterial A site and induce misreading of the genetic code. Point mutations of the ribosomal A site may confer resistance to aminoglycoside antibiotics. The influence of bacterial mutations (introduced by site-directed mutagenesis) on ribosomal drug susceptibility was investigated in vivo by determination of minimal inhibitory concentrations. To determine the origin of the various resistance phenotypes at a molecular level, the in vivo results were compared with the previously published crystal structures of paromomycin, tobramycin, and geneticin bound to oligonucleotides containing the minimal A site. Two regions appear crucial for binding in the A site: the single adenine residue at position 1408 and the non-Watson-Crick U1406.U1495 pair. The effects of mutations at those positions are modulated by the nature of the substituent at position 6' (either hydroxy or ammonium group) on ring I, by the number of positive charges on the antibiotic, and by the linkage between rings I and III (either 4,5 or 4,6). In particular, the analysis demonstrates: 1) that the C1409-G1491 to A1409-U1491 polymorphism (observed in 15 % of bacteria) is not associated with resistance, which indicates that it does not affect the stacking of ring I on residue 1491, 2) that the high-level resistance to 6'-NH3+ aminoglycosides exhibited by the A1408G mutation most probably results from the inability of ring I forming a pseudo base pair with G1408, which prevents its insertion inside the A site helix, and 3) that mutations of the uracil residues forming the U1406.U1495 pair either to cytosine or to adenine residues mostly confer low to moderate levels of drug resistance, whereas the U1406C/U1495A double mutation confers high-level resistance (except for neomycin), which suggests that aminoglycoside binding to the wild-type A site and its functional consequences strongly depend on a particular geometry of the U1406.U1495 pair. The relationships between the resistance phenotypes observed in vivo and the interactions described at the molecular level define the biological importance of the different structural interactions observed by X-ray crystallography studies.

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Peptide bond formation is the main catalytic function of the ribosome. The mechanism of catalysis is presumed to be highly conserved in all organisms. We tested the conservation by comparing mechanistic features of the peptidyl transfer reaction on ribosomes from Escherichia coli and the Gram-positive bacterium Mycobacterium smegmatis. In both cases, the major contribution to catalysis was the lowering of the activation entropy. The rate of peptide bond formation was pH independent with the natural substrate, aminoacyl-tRNA, but was slowed down 200-fold with decreasing pH when puromycin was used as a substrate analog. Mutation of the conserved base A2451 of 23 S rRNA to U did not abolish the pH dependence of the reaction with puromycin in M. smegmatis, suggesting that A2451 did not confer the pH dependence. However, the A2451U mutation alters the structure of the peptidyl transferase center and changes the pattern of pH-dependent rearrangements, as probed by chemical modification of 23 S rRNA. A2451 seems to function as a pivot point in ordering the structure of the peptidyl transferase center rather than taking part in chemical catalysis.Ribosomes catalyze peptide bond formation between aminoacyltRNA (aa-tRNA) 4 bound to the A site of the ribosome and peptidyltRNA at the P site. The active site for peptide bond formation, the peptidyl transferase center, is located on the large (50 S) ribosomal subunit. High-resolution crystal structures of the 50 S subunit have revealed that the peptidyl transferase center is composed of RNA (23 S rRNA), with no protein within 15 Å of the active site (1, 2). This implies that peptide bond formation is catalyzed by RNA and, thus, the ribosome is a ribozyme.The peptide bond is formed as a result of nucleophilic attack by the ␣-amino group of aa-tRNA on the ester carbonyl group of peptidyltRNA. The first step is the deprotonation of the ␣-NH 3 ϩ group to create the nucleophilic NH 2 group. The pK a of the ␣-NH 3 ϩ group in aa-tRNA is estimated to be around 8, and it is likely that the proton is accepted by water (3). Subsequent nucleophilic attack of the ␣-NH 2 group on the electrophilic carbonyl group leads to the formation of the zwitterionic tetrahedral intermediate, which, by deprotonation, forms the negatively charged tetrahedral intermediate. The breakdown of the tetrahedral intermediate is initiated by donating a proton back to the leaving oxygen to form the products, i.e. P-site deacylated tRNA and A-site peptidyl-tRNA.In principle, the ribosome may catalyze the reaction by several mechanisms, such as proper positioning of the peptidyl and aminoacyl ends of the tRNAs in the active site in a conformation suitable for the spontaneous reaction, general base-acid catalysis during deprotonation and protonation, or electrostatic stabilization of the transition state(s) (4 -9). The pH dependence of peptidyl transfer reaction catalyzed by Escherichia coli ribosomes suggested the importance of an ionizing group of the ribosome with a pK a of 7.5, which could contribute to chemical...

Self-diffusion coefficients of linear and cyclic alkanes in melt, in blends with equivalent linear alkanes, and dissolved in linear polyethylene, were measured by pulsed-gradient spin-echo nuclear magnetic resonance at various temperatures. The results indicate the following: (i) at the same carbon number, cyclic alkanes diffuse more slowly than linear alkanes in their respective melts, but linears and cyclics share a similar rapid rate of decrease with increasing carbon number; (ii) in blends of linear and cyclic alkanes at the same carbon number the single average diffusion coefficient observed varies monotonically as a function of composition; and (iii) two distinct diffusion coefficients are observed in the cycloalkane/linear polyethylene blends, with the extrapolated trace cycloalkane diffusion consistent with Rouse behavior. The results are compared with recent numerical simulations and with experiments in other polymer systems, forming a consistent picture of the effects of diffusant mass, molecular shape and flexibility, and the dynamic attributes of the host material.

SummaryThe methanogenic archaeon Methanobacterium thermoautotrophicum Marburg is infected by the doublestranded DNA phage ⌿M2. The complete phage genome sequence of 26 111 bp was established. Thirty-one open reading frames (orfs), all of them organized in the same direction of transcription, were identified. On the basis of comparison of the deduced amino acid sequences to known proteins and by searching for conserved motifs, putative functions were assigned to the products of six orfs. These included three proteins involved in packaging DNA into the capsid, two putative phage structural proteins and a protein related to the Int family of site-specific recombinases. Analysis of the N-terminal amino acid sequences of three phageencoded proteins led to the identification of two genes encoding structural proteins and of peiP, the structural gene of pseudomurein endoisopeptidase. This enzyme is involved in the lysis of host cells, and it appears to belong to a novel enzyme family. peiP was overexpressed in Escherichia coli, and its product was shown to catalyse the in vitro lysis of M. thermoautotrophicum cells. Comparison of the phage ⌿M2 DNA sequence with parts of the sequence of the wild-type phage ⌿M1 suggests that ⌿M2 is a deletion derivative, which formed by homologous recombination between two copies of a direct repeat.