Functional 70S hybrid ribosomes from blue-green algae and bacteria

Hybrid 70S ribosomes were produced by combining Anacystis nidulans and Escherichia coli 30S and 50S subunits. Both the A. nidulans 30S-E. coli 50S and E. coli 30S- A. nidulans 50S hybrids were functional in synthesizing protein when tested in a standard in vitro amino acid incorporating system. Both 70S hybrids were inhibited by streptomycin but the degree of inhibition was dependent upon the source of the 30S subunit. The ability to form functional 70S ribosomes from subunits of blue-green algae and bacteria is further evidence of the procaryotic nature of blue-greens and of the functional homology of the two protein synthesizing systems.     

RNA trafficking in axons

A substantial number of studies over a period of four decades have indicated that axons contain mRNAs and ribosomes, and are metabolically active in synthesizing proteins locally. For the most part, little attention has been paid to these findings until recently when the concept of targeting of specific mRNAs and translation in subcellular domains in polarized cells emerged to contribute to the likelihood and acceptance of mRNA targeting to axons as well. Trans-acting factor proteins bind to cis-acting sequences in the untranslated region of mRNAs integrated in ribonucleoprotein (RNPs) complexes determine its targeting in neurons. In vitro studies in immature axons have shown that molecular motors proteins (kinesins and myosins) associate to RNPs suggesting they would participate in its transport to growth cones. Tau and actin mRNAs are transported as RNPs, and targeted to axons as well as ribosomes. Periaxoplasmic ribosomal plaques (PARPs), which are systematically distributed discrete peripheral ribosome-containing, actin-rich formations in myelinated axons, also are enriched with actin and myosin Va mRNAs and additional regulatory proteins. The localization of mRNAs in PARPs probably means that PARPs are local centers of translational activity, and that these domains are the final destination in the axon compartment for targeted macromolecular traffic originating in the cell body. The role of glial cells as a potentially complementary source of axonal mRNAs and ribosomes is discussed in light of early reports and recent ultrastructural observations related to the possibility of glial-axon trans-endocytosis.     

The slow dissociation rate of K-1602 contributes to the enhanced inhibitory activity of this novel alkyl–aryl-bearing fluoroketolide

Ketolides belong to the latest generation of macrolides and are not only effective against macrolide susceptible bacterial strains but also against some macrolide resistant strains. Here we present data providing insights into the mechanism of action of K-1602, a novel alkyl–aryl-bearing fluoroketolide. According to our data, the K-1602 interacts with the ribosome as a one-step slow binding inhibitor, displaying an association rate constant equal to 0.28?×?10^4?M^?1 s^?1 and a dissociation rate constant equal to 0.0025?min^?1. Both constants contribute to produce an overall inhibition constant K_i equal to 1.49?×?10^?8?M, which correlates very well with the superior activity of this compound when compared with many other ketolides or fluoroketolides.     

RNase MRP and rRNA processing

RNase MRP is a ribonucleoprotein enzyme with a structure similar to RNase P. It is required for normal processing of precursor rRNA, cleaving it in the Internal Transcribed Spacer 1. Abbreviations: RNase MRP RNase for mitochondrial RNA processing; also involved in pre-rRNA processing; RNase P - RNase for pre-tRNA processing; snoRNA - small nucleolar RNA; RNP - RNA-protein particle; snoRNP - small nucleolar RNA-protein particle.     

Ribosomal preparations may induce maturation in Xenopus laevis oocytes

Xenopus laevis oocytes undergo maturation when they are injected with large quantities of crude ribosomes from various origins: X laevis full-grown or matured oocytes, Xenopus ovaries and embryos, Xenopus liver or mouse liver. All have the same efficiency, whatever their origin: they include 50-90% maturation in the injected oocytes at about the same speed as progesterone treatment. The ribosomal preparations are inactive wen injected into recipient oocytes pretreated with cholera toxin or cycloheximide. After dissociation with the high salt extract, but not with the subunits. Hypotheses concernning the mode action of this ribosomal extract are disussed.     

Polysome Fractionation and Analysis of Mammalian Translatomes on a Genome-wide Scale

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. Accordingly, dysregulation of mRNA translation is considered to play a major role in a variety of pathological states including cancer. Ribosomes also host chaperones, which facilitate folding of nascent polypeptides, thereby modulating function and stability of newly synthesized polypeptides. In addition, emerging data indicate that ribosomes serve as a platform for a repertoire of signaling molecules, which are implicated in a variety of post-translational modifications of newly synthesized polypeptides as they emerge from the ribosome, and/or components of translational machinery. Herein, a well-established method of ribosome fractionation using sucrose density gradient centrifugation is described. In conjunction with the in-house developed “anota” algorithm this method allows direct determination of differential translation of individual mRNAs on a genome-wide scale. Moreover, this versatile protocol can be used for a variety of biochemical studies aiming to dissect the function of ribosome-associated protein complexes, including those that play a central role in folding and degradation of newly synthesized polypeptides.     

Expansion of the Genetic Code Through the Use of Modified Bacterial Ribosomes

Biological protein synthesis is mediated by the ribosome, and employs ~20 proteinogenic amino acids as building blocks. Through the use of misacylated tRNAs, presently accessible by any of several strategies, it is now possible to employ in vitro and in vivo protein biosynthesis to elaborate proteins containing a much larger variety of amino acid building blocks. However, the incorporation of this broader variety of amino acids is limited to those species utilized by the ribosome. As a consequence, virtually all of the substrates utilized over time have been L-α-amino acids. In recent years, a variety of structural and biochemical studies have provided important insights into those regions of the 23S ribosomal RNA that are involved in peptide bond formation. Subsequent experiments, involving the randomization of key regions of 23S rRNA required for peptide bond formation, have afforded libraries of E. coli harboring plasmids with the rrnB gene modified in the key regions. Selections based on the use of modified puromycin derivatives with altered amino acids then identified clones uniquely sensitive to individual puromycin derivatives. These clones often recognized misacylated tRNAs containing altered amino acids similar to those in the modified puromycins, and incorporated the amino acid analogues into proteins. In this fashion, it has been possible to realize the synthesis of proteins containing D-amino acids, β-amino acids, phosphorylated amino acids, as well as long chain and cyclic amino acids in which the nucleophilic amino group is not in the α-position. Of special interest have been dipeptides and dipeptidomimetics of diverse utility.     

The interaction between streptomycin and ribosomal RNA

The present study shows that a mutation in the 530 loop of 16S rRNA impairs the binding of streptomycin to the bacterial ribosome, thereby restricting the misreading effect of the drug. Previous reports demonstrated that proteins S4, S5 and S12 as well as the 915 region of 16S rRNA are involved in the binding of streptomycin, and indicated that the drug not only interacts with the 30S subunit but also with the 50S subunit. The relationship between the target of streptomycin and its known interference with the proofreading control of translational accuracy is examined in light of these results.