Philippe Dessen

Kinetics of the reversible association of ribosomal subunits: stopped-flow studies of the rate law and of the effect of Mg2+.

Abstract The kinetics of association and dissociation of Escherichia coli 30 S and 50 S ribosomal subunits appear to fit the simple scheme over a wide range of Mg 2+ and ribosome concentrations, for the preparations studied (which have a sharp [Mg 2+ ]-dependence on the equilibrium degree of association, e.g. 10% to 90% for 1.5 m m to 3.5 m m ). Both rate constants depend strongly upon magnesium ion concentration ( k 2 goes from 0.04 × 10 6 to 21 × 10 6 m −1 s −1 as [Mg 2+ ] goes from 1.5 m m to 8 m m ; k 1 goes from 150 to 2 s −1 in the interval 1.0 to 3.0 m m ), but k 1 may level off above 3.0 m m and k 2 increases slowly at high [Mg 2+ ]. (The highest rate may not be far from the diffusion-controlled limit.) The primary effect of Mg 2+ , as calculated from the rather large changes in binding as a function of [Mg 2+ ], is to decrease the contribution of electrostatic repulsion to the free energy of activation; specific, or class-specific, interactions of di- and multivalent cations with unknown ribosomal substructures may modulate this effect.

The molecular weight and subunit composition of phenylalanyl-tRNA synthetase from Escherichia coli K-12

Summary Phenylalanyl tRNA synthetase from Escherichia coli K-12 (strain CA 244) was purified by a procedure which differs from that developed by others for the isolation of the enzyme from strains B and K-10. The purified enzyme appears homogenous on sedimentation equilibrium analysis and gel electrophoresis. Its specific activity in the aminoacylation of tRNA is identical to that of the enzyme from strain K-10. However, its molecular weight and subunit structure differ radically from those reported earlier for the enzymes from strains B and K-10. The molecular weight is 240,000 based on equilibrium sedimentation and light scattering measurements and 270,000 as deduced from Sephadex G200 gel filtration and gel electrophoresis of the subunits in the presence of sodium dodecyl sulfate. The results are consistent with a tetrameric structure of the type α2β2, composed of two kinds of subunits with molecular weights of 37,000 and 98,000, respectively.

[11] Properties and specificity of methionyl-tRNAfMet formyltransferase from Escherichia coli

Publisher Summary This chapter describes large-scale procedure for the purification of Escherichia coli methionyl-tRNA Met f formyltransferase to homogeneity. It also presents the studies with unformylatable tRNAs. Enzymatic activity of formyltransferase can be followed by formylating L- methionyl-tRNA Met f with 10-[ 14 C]formyltetrahydrofolate and measuring the incorporation of radioactivity into material insoluble in trichloroacetic acid. More conveniently, it can be followed by measuring the resistance conferred by formylation against deacylation of L- [ 14 C] methionyl-tRNA Met f . The final volume of the assay mixture is made 100 μ l. The purification method requires about 4-5 weeks for the isolation of ∼20 mg homogeneous formyltransferase excluding the time for the preparation of the cells. The purified preparations of methionyl-tRNA Met f formyltransferase are stable for months when kept in 20 m M imidazole-HCl, 0.1 m M EDTA, 10 m M 2-mercaptoethanol containing 50% glycerol, and 150 m M KCl at -15°. Also in the absence of added ionic strength, the formyltransferase activity decreases within 10 min, reaching a plateau value depending on the initial concentration of the enzyme and the surface of glass in contact with the enzyme.

Initiation of Protein Synthesis in Prokaryotes

Initiation of protein synthesis appears to be a process of great complexity in both prokaryotic and eukaryotic systems. A possible reason for such complexity, particularly in comparison with the process of elongation of nascent proteins, may reside in gene regulation operating through the selection of mRNA or cistrons by ribosomes and protein factors. It is known that in prokaryotes the main control occurs at the transcriptional level. However, for polycistronic mRNA’s there are a few facts consistent with a regulation at the translational level, and this is particularly shown in the case of RNA phage infection of E. coli (MS2, Qs, R17) where the three cistrons of the phage RNA are translated with different frequencies in vivo [1] although they are present in equal concentrations. Therefore, it is conceivable that a control exists at the translational level which might have a specific function in fine regulation of bacterial protein synthesis.

Transcriptional response to hypoxic stress in melanoma and prognostic potential of GBE1 and BNIP3

Gradients of hypoxia occur in most solid tumors and cells found in hypoxic regions are associated with the most aggressive and therapy-resistant fractions of the tumor. Despite the ubiquity and importance of hypoxia responses, little is known about the variation in the global transcriptional response to hypoxia in melanoma. Using microarray technology, whole genome gene expression profiling was first performed on established melanoma cell lines. From gene set enrichment analyses, we derived a robust 35 probes signature (hypomel for HYPOxia MELanoma) associated with hypoxia-response pathways, including 26 genes up regulated, and 9 genes down regulated. The microarray data were validated by RT-qPCR for the 35 transcripts. We then validated the signature in hypoxic zones from 8 patient specimens using laser microdissection or macrodissection of Formalin fixed-paraffin-embedded (FFPE) material, followed with RT-qPCR. Moreover, a similar hypoxia-associated gene expression profile was observed using NanoString technology to analyze RNAs from FFPE melanoma tissues of a cohort of 19 patients treated with anti-PD1. Analysis of NanoString data from validation sets using Non-Negative Matrix Factorization (NMF) analysis (26 genes up regulated in hypoxia) and dual clustering (samples and genes) further revealed that the increased level of BNIP3 (Bcl-2 adenovirus E1B 19 kDa-interacting protein 3)/GBE1 (glycogen branching enzyme1) differential pair correlates with the lack of response of melanoma patients to anti-PD1 (pembrolizumab) immunotherapy. These studies suggest that through elevated glycogenic flux and induction of autophagy, hypoxia is a critical molecular program that could be considered as a prognostic factor for melanoma.

Methionyl-tRNA Synthetase from Escherichia coli: Structure-Function Relationships of a Dimeric Enzyme with Repeated Sequences

Aminoacyl-tRNA synthetases display a variety of molecular structures. For instance, in Escherichia coli the molecular weights of native aminoacyl-tRNA synthetases range from 44,000 in the case of cysteinyl-tRNA synthetase to 265,000 in the case of phenylalanyl-tRNA synthetase (Fayat et al. 1974). Moreover, depending on the enzyme considered, the synthetases consist of a single polypeptide chain or result from the assembly of identical or nonidentical subunits. Structures of the α , α 2 , and α 2 β 2 types can be distinguished (for a review, see Ofengand 1977). Until recently, this complex pattern of structures has defied any attempt to find a unifying trait within this class of isofunctional enzymes. In the past few years it has been shown that several aminoacyl-tRNA synthetases exhibit extensive sequence duplication inside polypeptide chains. Those enzymes that have so far been shown to possess this property are composed of one or more 100,000-m.w. polypeptide chain. On the basis of this observation, the bacterial aminoacyl-tRNA synthetases can be schematically grouped into a minimum of four nonexclusive classes based on protomer number and degree of sequence duplication. The first class groups the enzymes composed of two identical subunits that do not have internal repeats. The molecular weights of the protomers are in the range of 35,000–55,000. The second class are monomeric enzymes of about 100,000 m.w. with extensive duplication (Kula 1973; Koch et al. 1974; Waterson and Konigsberg 1974; Bruton 1975). The third class is represented by the bacterial methionyl-tRNA synthetase. This enzyme is a dimer (2 × 85,000) composed of...