Dial tm for rescue: tmRNA engages ribosomes stalled on defective mRNAs

Ribosomes translate genetic information encoded by mRNAs into protein chains with high fidelity. Truncated mRNAs lacking a stop codon will cause the synthesis of incomplete peptide chains and stall translating ribosomes. In bacteria, a ribonucleoprotein complex composed of tmRNA, a molecule that combines the functions of tRNAs and mRNAs, and small protein B (SmpB) rescues stalled ribosomes. The SmpB-tmRNA complex binds to the stalled ribosome, allowing translation to resume at a short internal tmRNA open reading frame that encodes a protein degradation tag. The aberrant protein is released when the ribosome reaches the stop codon at the end of the tmRNA open reading frame and the fused peptide tag targets it for degradation by cellular proteases. The recently determined NMR structures of SmpB, the crystal structure of the SmpB-tmRNA complex and the cryo-EM structure of the SmpB-tmRNA-EF-Tu-ribosome complex have provided first detailed insights into the intricate mechanisms involved in ribosome rescue.     

Identification of dominant negative mutants of Rheb GTPase and their use to implicate the involvement of human Rheb in the activation of p70S6K

Rheb GTPases represent a unique family of the Ras superfamily of G-proteins. Studies on Rheb in Schizosaccharomyces pombe and Drosophila have shown that this small GTPase is essential and is involved in cell growth and cell cycle progression. The Drosophila studies also raised the possibility that Rheb is involved in the TOR/S6K signaling pathway. In this paper, we first report identification of dominant negative mutants of S. pombe Rheb (SpRheb). Screens of a randomly mutagenized SpRheb library yielded a mutant, SpRhebD60V, whose expression in S. pombe results in growth inhibition, G1 arrest, and induction of fnx1+, a gene whose expression is induced by the disruption of Rheb. Alteration of the Asp-60 residue to all possible amino acids by site-directed mutagenesis led to the identification of two particularly strong dominant negative mutants, D60I and D60K. Characterization of these dominant negative mutant proteins revealed that D60V and D60I exhibit preferential binding of GDP, while D60K lost the ability to bind both GTP and GDP. A possible use of the dominant negative mutants in the study of mammalian Rheb was explored by introducing dominant negative mutations into human Rheb. We show that transient expression of the wild type Rheb1 or Rheb2 causes activation of p70S6K, while expression of Rheb1D60K mutant results in inhibition of basal level activity of p70S6K. In addition, Rheb1D60K and Rheb1D60V mutants blocked nutrient- or serum-induced activation of p70S6K. This provides critical evidence that Rheb plays a role in the mTOR/S6K pathway in mammalian cells.     

Peptidoglycan structure of Lactobacillus casei, a species highly resistant to glycopeptide antibiotics.

The structure of the peptidoglycan of Lactobacillus casei ATCC 393, a species highly resistant to glycopeptide antibiotics, was examined. After digestion, 23 muropeptides were identified; monomers represented 44.7% of all muropeptides, with monomer tetrapeptides being the major ones. Fifty-nine percent of the peptidoglycan was O-acetylated. The cross-bridge between D-alanine and L-lysine consisted of one asparagine, although aspartate could be found in minor quantities. Since UDP-MurNAc-tetrapeptide-D-lactate is the normal cytoplasmic precursor found in this species, monomer tetrapeptide-lactate was expected to be found. However, such a monomer was found only after exposure to penicillin, suggesting that penicillin-sensitive D,D-carboxypeptidases were very active in normal growing cells.     

Genetically Related Human Immunodeficiency Virus Type 1 in Three Adults of a Family with No Identified Risk Factor for Intrafamilial Transmission

A small number of cases of human immunodeficiency virus (HIV) infection have been reported in individuals with no identified risk factors for transmission. We report on the seroconversion of the 61-year-old mother and the subsequent finding of HIV seropositivity in the 66-year-old father of a 31-year-old AIDS patient. Extensive investigation failed to identify any risk factor for intrafamilial transmission. We conducted a genetic analysis and determined the amino acid signature patterns of the V3, V4, and V5 hypervariable domains and flanking regions in the HIV-1 gp120 env gene of 26 clones derived from proviral DNA in peripheral blood mononuclear cells of the members of the family. env sequences of the viruses isolated from the patients were compared with sequences of HIV-1 subtype B viruses from Europe and local field isolates. Phylogenetic analysis revealed that the sequences of the viruses isolated from the patients were genetically related and formed an intrafamilial cluster of HIV-1 distinct from other subtype B viruses. Interindividual nucleotide variability in the C2-V3 and V4-C4-V5 domains ranged between 1.2 and 5.0% and between 2.2 and 7.5%, respectively, whereas divergence between HIV strains from the patients and control viral strains ranged from 6.6 to 29.3%. The amino acid signature patterns of viral clones from the three patients were closely related. In the C2-V3 region, two minor clones derived from the son’s virus showed less nucleotide divergence (mean, 3.5 and 3.9%) than did the clones derived from the viruses of both parents or the seven other predominant clones derived from the virus from the son (mean, 5.4%). The top of the V3 loop of the last two clones and of all viral clones from the parents exhibited an unusual GPGG sequence. This is the first report of genotypic relatedness of HIV-1 in three adults of the same family in the absence of identified risk factor for transmission between the members of the family. Our findings suggest that atypical transmission of HIV may occur.     

The path integral for dendritic trees

We construct the path integral for determining the potential on any dendritic tree described by a linear cable equation. This is done by generalizing Brownian motion from a line to a tree. We also construct the path integral for dendritic structures with spatially-varying and/or time-dependent membrane conductivities due, for example, to synaptic inputs. The path integral allows novel computational techniques to be applied to cable problems. Our anlaysis leads ultimately to an exact expression for the Green's function on a dendritic tree of arbitrary geometry expressed in terms of a set of simple diagrammatic rules. These rules providing a fast and efficient method for solving complex cable problems.Research supported by Department of Energy Contracts DE-AC02-76ER03230 and DE-AC02-76ER03069 and by National Institute of Mental Health grant MH46742     

A phasmid shuttle vector for the cloning of complex operons in Salmonella

Phasmid (phage plasmid hybrid) P4 vir1 can be propagated in Escherichia coli as a helper-dependent lytic phage, as a plasmid, or as a prophage. On the basis of an understanding of these modes of propagation, derivatives of P4 have been constructed for use as cloning vectors. In this report we demonstrate that phasmid P4 (i) will propagate as a helper-dependent lytic phage and as a plasmid in Salmonella spp. and (ii) can be used as a high efficiency phage shuttle vector for the reversible transfer of cloned genes between Salmonella spp. and E. coli. For both E. coli and Salmonella spp., P4 phage-mediated gene transfer proved to be only 10-fold lower than plaquing efficiency. For the case of Salmonella spp., this frequency is ca. 10(4)-fold more efficient than is typically found for the transformation of DNA molecules. The usefulness of this cloning vector system for analyses of pathogenic virulence factors is demonstrated by the cloning and expression of both the P pilus adhesin operon and the hemolysin operon of uropathogenic E. coli.     

A Conserved Circadian Function for the Neurofibromatosis 1 Gene

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Peptidoglycan synthesis and structure in Staphylococcus haemolyticus expressing increasing levels of resistance to glycopeptide antibiotics.

The structures of cytoplasmic peptidoglycan precursor and mature peptidoglycan of an isogenic series of Staphylococcus haemolyticus strains expressing increasing levels of resistance to the glycopeptide antibiotics teicoplanin and vancomycin (MICs, 8 to 32 and 4 to 16 microg/ml, respectively) were determined. High-performance liquid chromatography, mass spectrometry, amino acid analysis, digestion by R39 D,D-carboxypeptidase, and N-terminal amino acid sequencing were utilized. UDP-muramyl-tetrapeptide-D-lactate constituted 1.7% of total cytoplasmic peptidoglycan precursors in the most resistant strain. It is not clear if this amount of depsipeptide precursor can account for the levels of resistance achieved by this strain. Detailed structural analysis of mature peptidoglycan, examined for the first time for this species, revealed that the peptidoglycan of these strains, like that of other staphylococci, is highly cross-linked and is composed of a lysine muropeptide acceptor containing a substitution at its epsilon-amino position of a glycine-containing cross bridge to the D-Ala 4 of the donor, with disaccharide-pentapeptide frequently serving as an acceptor for transpeptidation. The predominant cross bridges were found to be COOH-Gly-Gly-Ser-Gly-Gly-NH2 and COOH-Ala-Gly-Ser-Gly-Gly-NH2. Liquid chromatography-mass spectrometry analysis of the peptidoglycan of resistant strains revealed polymeric muropeptides bearing cross bridges containing an additional serine in place of glycine (probable structures, COOH-Gly-Ser-Ser-Gly-Gly-NH2 and COOH-Ala-Gly-Ser-Ser-Gly-NH2). Muropeptides bearing an additional serine in their cross bridges are estimated to account for 13.6% of peptidoglycan analyzed from resistant strains of S. haemolyticus. A soluble glycopeptide target (L-Ala-gamma-D-iso-glutamyl-L-Lys-D-Ala-D-Ala) was able to more effectively compete for vancomycin when assayed in the presence of resistant cells than when assayed in the presence of susceptible cells, suggesting that some of the resistance was directed towards the cooperativity of glycopeptide binding to its target. These results are consistent with a hypothesis that alterations at the level of the cross bridge might interfere with the binding of glycopeptide dimers and therefore with the cooperative binding of the antibiotic to its target in situ. Glycopeptide resistance in S. haemolyticus may be multifactorial.