Presence Of Ribosomal Proteins Research Articles

RsmG forms stable complexes with premature small subunit rRNA during bacterial ribosome biogenesis.

The ribosome is the ribonucleoprotein machine that carries out protein biosynthesis in all forms of life. Perfect synchronization between ribosomal RNA (rRNA) transcription, folding, post-transcriptional modification, maturation, and assembly of r-proteins is essential for the rapid formation of structurally and functionally accurate ribosomes. Many RNA nucleotide modification enzymes may function as assembly factors that oversee the accuracy of ribosome assembly. The protein RsmG is a methyltransferase enzyme that is responsible for N7 methylation in G527 of 16S rRNA. Here we illustrate the ability of RsmG to bind various premature small subunit ribosomal RNAs with contrasting affinities. Protein RsmG binds with approximately 15-times higher affinity to premature 16S rRNA with the full leader sequence compared to that of mature 16S rRNA. Various r-proteins which bind to the 5′-domain influence RsmG binding. The observed binding cooperativity between RsmG and r-proteins is sensitive to the maturation status of premature small subunit rRNA. However, neither the maturation of 16S rRNA nor the presence of various r-proteins significantly influence the methylation activity of RsmG. The capability of RsmG to bind to premature small subunit rRNA and alter its binding preference to various RNA–protein complexes based on the maturation of rRNA indicates its ability to influence ribosome assembly.

Translation initiation in bacterial polysomes through ribosome loading on a standby site on a highly translated mRNA

During translation, consecutive ribosomes load on an mRNA and form a polysome. The first ribosome binds to a single-stranded mRNA region and moves toward the start codon, unwinding potential mRNA structures on the way. In contrast, the following ribosomes can dock at the start codon only when the first ribosome has vacated the initiation site. Here we show that loading of the second ribosome on a natural 38-nt-long 5' untranslated region of lpp mRNA, which codes for the outer membrane lipoprotein from Escherichia coli, takes place before the leading ribosome has moved away from the start codon. The rapid formation of this standby complex depends on the presence of ribosomal proteins S1/S2 in the leading ribosome. The early recruitment of the second ribosome to the standby site before translation by the leading ribosome and the tight coupling between translation elongation by the first ribosome and the accommodation of the second ribosome can contribute to high translational efficiency of the lpp mRNA.

Intact spore MALDI-TOF mass spectrometry and proteomic analysis of Puccinia pathogenic fungi

The aim of this work was to develop a method for the identification of pathogens causing rust diseases of crops using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) of intact cells or spores (IC/IS). All optimizations were performed with Puccinia triticina, the causal agent of wheat leaf rust. Experiments included selection of washing solvents for spores, finding of an optimal concentration of spores in suspension and the most suitable matrix system as well as an evaluation of different sample preparation techniques. The best results were obtained when the spores were washed with acetonitrile/0.1% (v/v) trifluoroacetic acid, 7:3, v/v. A mixture of ferulic and sinapinic acids (5:15mgml−1) dissolved in acetonitrile/2.5% (v/v) trifluoroacetic acid, 7:3, v/v, was found optimal for the deposition of samples (50μg spores per μl) by two-layer volume technique. The optimized protocol was subsequently applied to other Puccinia species (Puccinia graminis, Puccinia striiformis and Puccinia coronata). Together with the use of the software BIOSPEAN, not only different species but also various pathotypes of the same species, which differ in their virulence, could be discriminated. There were 108 and 29 proteins identified from P. striiformis and P. graminis spores, respectively, after an acidic extraction in the matrix solvent mimicking the sample preparation for MALDI. Besides the presence of ribosomal proteins, histones, regulatory proteins and enzymes, also extracellular proteins participating in the pathogenesis were found. Finally, for both species, several proteins were assigned to signals in typical mass spectrometric profiles and suggested as diagnostic markers.

Subribosomal particle analysis reveals the stages of bacterial ribosome assembly at which rRNA nucleotides are modified.

Modified nucleosides of ribosomal RNA are synthesized during ribosome assembly. In bacteria, each modification is made by a specialized enzyme. In vitro studies have shown that some enzymes need the presence of ribosomal proteins while other enzymes can modify only protein-free rRNA. We have analyzed the addition of modified nucleosides to rRNA during ribosome assembly. Accumulation of incompletely assembled ribosomal particles (25S, 35S, and 45S) was induced by chloramphenicol or erythromycin in an exponentially growing Escherichia coli culture. Incompletely assembled ribosomal particles were isolated from drug-treated and free 30S and 50S subunits and mature 70S ribosomes from untreated cells. Nucleosides of 16S and 23S rRNA were prepared and analyzed by reverse-phase, high-performance liquid chromatography (HPLC). Pseudouridines were identified by the chemical modification/primer extension method. Based on the results, the rRNA modifications were divided into three major groups: early, intermediate, and late assembly specific modifications. Seven out of 11 modified nucleosides of 16S rRNA were late assembly specific. In contrast, 16 out of 25 modified nucleosides of 23S rRNA were made during early steps of ribosome assembly. Free subunits of exponentially growing bacteria contain undermodified rRNA, indicating that a specific set of modifications is synthesized during very late steps of ribosome subunit assembly.

RNA—RNA and RNA—protein interactions in 30 S ribosomal subunits Association of 16 S rRNA fragments in the presence of ribosomal proteins

The E. coli 16 S rRNA with single-site breaks centered at position 777 or 785 was obtained by R Nase H site-specific cleavage of rRNA. Spontaneous dissociation of the cleaved 16 S rRNA into fragments occurred under ‘native’ conditions. The reassociation of the 16 S rRNA fragments was possible only in the presence of ribosomal proteins. The combination of S4 and S16(S17) ribosomal proteins interacting mainly with the 5′-end domain of 16 S rRNA was sufficient for reassociation of the fragments. The 30 S subunits with fragmented RNA at ca. 777 region retained some poly(U)-directed protein synthetic activity.

Electrostatic potential of macromolecules measured by pKa shift of a fluorophore. 1. The 3' terminus of 16s RNA

We have investigated the use of the pH-sensitive fluorescein label as a probe for electrostatic potential in macromolecules. The practicality of this technique is demonstrated by its application to the 16S RNA molecule. The dependence of the electrostatic potential upon ionic conditions and upon the presence of ribosomal proteins and the state of the RNA was studied. The combination of electrostatic and anisotropy data emphasizes the rôle of the 30S ribosomal proteins, rather than of the renaturation of the 16S RNA or the presence of the 50S subunit, in shaping the environment of the 3' terminus of the 16S RNA in the active ribosome.

Ribosomal proteins: XLIII. In vivo assembly of Escherichia coli ribosomal proteins

Isolation of ribosomal precursors from Escherichia coli K12 is described. The RNA and protein content of the precursor particles was determined. One physiologically stable precursor was found for the 30 S subunit. The assembly scheme is as follows: p16 S RNA + 9 proteins → p30 S (“21 S” precursor) p30 S + 12 proteins → 30 S subunit where p is precursor. Each of the two precursors for the 50 S subunit, P 150 S and p 250 S (“32 S” and “43 S” precursors, respectively), contains p5 S + p23 S RNA's in a 1:1 molar ratio. The assembly scheme is as follows: p23 S RNA + p5 S RNA + 16 or 17 proteins → p 150 S ▪ In contrast to the p 250 S precursor the p 150 S precursor is not similar to any core particles, which were obtained by treating 50 S subunits with different concentrations of LiCl or CsCl. The precursors p30 S and p 250 S can be converted into active 30 S and 50 S sub-units, respectively, by incubation at 42 °C in the presence of ribosomal proteins and under RNA methylating conditions.