Ilya A. Osterman

Comparison of mRNA features affecting translation initiation and reinitiation

Regulation of gene expression at the level of translation accounts for up to three orders of magnitude in its efficiency. We systematically compared the impact of several mRNA features on translation initiation at the first gene in an operon with those for the second gene. Experiments were done in a system with internal control based on dual cerulean and red (CER/RFP) fluorescent proteins. We demonstrated significant differences in the efficiency of Shine Dalgarno sequences acting at the leading gene and at the following genes in an operon. The majority of frequent intercistronic arrangements possess medium SD dependence, medium dependence on the preceding cistron translation and efficient stimulation by A/U-rich sequences. The second cistron starting immediately after preceding cistron stop codon displays unusually high dependence on the SD sequence.

The last rRNA methyltransferase of E. coli revealed: The yhiR gene encodes adenine-N6 methyltransferase specific for modification of A2030 of 23S ribosomal RNA

The ribosomal RNA (rRNA) of Escherichia coli contains 24 methylated residues. A set of 22 methyltransferases responsible for modification of 23 residues has been described previously. Herein we report the identification of the yhiR gene as encoding the enzyme that modifies the 23S rRNA nucleotide A2030, the last methylated rRNA nucleotide whose modification enzyme was not known. YhiR prefers protein-free 23S rRNA to ribonucleoprotein particles containing only part of the 50S subunit proteins and does not methylate the assembled 50S subunit. We suggest renaming the yhiR gene to rlmJ according to the rRNA methyltransferase nomenclature. The phenotype of yhiR knockout gene is very mild under various growth conditions and at the stationary phase, except for a small growth advantage at anaerobic conditions. Only minor changes in the total E. coli proteome could be observed in a cell devoid of the 23S rRNA nucleotide A2030 methylation.

Attenuation-Based Dual-Fluorescent-Protein Reporter for Screening Translation Inhibitors

ABSTRACT A reporter construct was created on the basis of the transcription attenuator region of the Escherichia coli tryptophan operon. Dual-fluorescent-protein genes for red fluorescent protein and cerulean fluorescent protein were used as a sensor and internal control of gene expression. The sequence of the attenuator was modified to avoid tryptophan sensitivity while preserving sensitivity to ribosome stalling. Antimicrobial compounds which cause translation arrest at the stage of elongation induce the reporter both in liquid culture and on an agar plate. This reporter could be used for high-throughput screening of translation inhibitors.

Modifications of ribosomal RNA: From enzymes to function

Modified nucleosides are present in all kinds of stable RNA molecules, tRNAs being particularly rich in them (Auffinger and Westhof, 1998). Ribosomal RNA (rRNA) from all organisms contains modifications, and there is a correlation between the overall complexity of an organism and the number of modified nucleosides in its rRNA. The rRNA of the most primitive bacteria, such as some Mycoplasma species, may possess only 14 modified nucleosides (de Crecy-Lagard et al., 2007). In Escherichia coli, there are 36 modified nucleosides in rRNA (Table I). Yeast ribosomes possess about one hundred rRNA modifications, human rRNA over two hundred (Ofengand and Fournier, 1998; Decatur and Fournier, 2002). Eukaryotes and archaea use snoRNA guided rRNA modification mechanism. This mechanism allows archaea and eukarya to use a limited number of modification enzymes, mainly pseudouridine synthase and 2′-O-methyltransferase to introduce the majority of their rRNA modifications (Decatur and Fournier, 2002). By contrast, bacteria have developed specific enzymes for each one of the (fewer) modifications they have. Nevertheless, there are many different rRNA modifications in bacteria. Despite intensive study for several decades, many open questions remain regarding the functional role of modified rRNA nucleosides. In this review we will focus on rRNA modifications in E. coli and discuss their possible functions.

How much can we learn about the function of bacterial rRNA modification by mining large-scale experimental datasets?

Modification of ribosomal RNA is ubiquitous among living organisms. Its functional role is well established for only a limited number of modified nucleotides. There are examples of rRNA modification involvement in the gene expression regulation in the cell. There is a need for large data set analysis in the search for potential functional partners for rRNA modification. In this study, we extracted phylogenetic profile, genome neighbourhood, co-expression and phenotype profile and co-purification data regarding Escherichia coli rRNA modification enzymes from public databases. Results were visualized as graphs using Cytoscape and analysed. Majority linked genes/proteins belong to translation apparatus. Among co-purification partners of rRNA modification enzymes are several candidates for experimental validation. Phylogenetic profiling revealed links of pseudouridine synthetases with RF2, RsmH with translation factors IF2, RF1 and LepA and RlmM with RdgC. Genome neighbourhood connections revealed several putative functionally linked genes, e.g. rlmH with genes coding for cell wall biosynthetic proteins and others. Comparative analysis of expression profiles (Gene Expression Omnibus) revealed two main associations, a group of genes expressed during fast growth and association of rrmJ with heat shock genes. This study might be used as a roadmap for further experimental verification of predicted functional interactions.

Amicoumacin A induces cancer cell death by targeting the eukaryotic ribosome

Amicoumacin A is an antibiotic that was recently shown to target bacterial ribosomes. It affects translocation and provides an additional contact interface between the ribosomal RNA and mRNA. The binding site of amicoumacin A is formed by universally conserved nucleotides of rRNA. In this work, we showed that amicoumacin A inhibits translation in yeast and mammalian systems by affecting translation elongation. We determined the structure of the amicoumacin A complex with yeast ribosomes at a resolution of 3.1  Å. Toxicity measurement demonstrated that human cancer cell lines are more susceptible to the inhibition by this compound as compared to non-cancerous ones. This might be used as a starting point to develop amicoumacin A derivatives with clinical value.

Methylated 23S rRNA nucleotide m2G1835 of Escherichia coli ribosome facilitates subunit association

Among 4.5 thousand nucleotides of Escherichia coli ribosome 36 are modified. These nucleotides are clustered in the functional centers of ribosome, particularly on the interface of large and small subunits. Nucleotide m(2)G1835 located on the 50S side of intersubunit bridge cluster B2 is modified by N2-methyltransferase RlmG. By means of isothermal titration calorimetry and Rayleigh light scattering, we have found that methylation of m(2)G1835 specifically enhances association of ribosomal subunits. No defects in fidelity of translation or interaction with translation GTPases could be ascribed to the ribosomes unmethylated at G1835 of the 23S rRNA. Methylation of G1835 was found to provide a significant advantage for bacteria at osmotic and oxidative stress.

Regulation of Flagellar Gene Expression in Bacteria.

The flagellum of a bacterium is a supramolecular structure of extreme complexity comprising simultaneously both a unique system of protein transport and a molecular machine that enables the bacterial cell movement. The cascade of expression of genes encoding flagellar components is closely coordinated with the steps of molecular machine assembly, constituting an amazing regulatory system. Data on structure, assembly, and regulation of flagellar gene expression are summarized in this review. The regulatory mechanisms and correlation of the process of regulation of gene expression and flagellum assembly known from the literature are described.

Sorting Out Antibiotics' Mechanisms of Action: a Double Fluorescent Protein Reporter for High-Throughput Screening of Ribosome and DNA Biosynthesis Inhibitors.

ABSTRACT In order to accelerate drug discovery, a simple, reliable, and cost-effective system for high-throughput identification of a potential antibiotic mechanism of action is required. To facilitate such screening of new antibiotics, we created a double-reporter system for not only antimicrobial activity detection but also simultaneous sorting of potential antimicrobials into those that cause ribosome stalling and those that induce the SOS response due to DNA damage. In this reporter system, the red fluorescent protein gene rfp was placed under the control of the SOS-inducible sulA promoter. The gene of the far-red fluorescent protein, katushka2S, was inserted downstream of the tryptophan attenuator in which two tryptophan codons were replaced by alanine codons, with simultaneous replacement of the complementary part of the attenuator to preserve the ability to form secondary structures that influence transcription termination. This genetically modified attenuator makes possible Katushka2S expression only upon exposure to ribosome-stalling compounds. The application of red and far-red fluorescent proteins provides a high signal-to-background ratio without any need of enzymatic substrates for detection of the reporter activity. This reporter was shown to be efficient in high-throughput screening of both synthetic and natural chemicals.

Application of sorting and next generation sequencing to study 5′-UTR influence on translation efficiency in Escherichia coli

Abstract Yield of protein per translated mRNA may vary by four orders of magnitude. Many studies analyzed the influence of mRNA features on the translation yield. However, a detailed understanding of how mRNA sequence determines its propensity to be translated is still missing. Here, we constructed a set of reporter plasmid libraries encoding CER fluorescent protein preceded by randomized 5΄ untranslated regions (5΄-UTR) and Red fluorescent protein (RFP) used as an internal control. Each library was transformed into Escherchia coli cells, separated by efficiency of CER mRNA translation by a cell sorter and subjected to next generation sequencing. We tested efficiency of translation of the CER gene preceded by each of 48 natural 5΄-UTR sequences and introduced random and designed mutations into natural and artificially selected 5΄-UTRs. Several distinct properties could be ascribed to a group of 5΄-UTRs most efficient in translation. In addition to known ones, several previously unrecognized features that contribute to the translation enhancement were found, such as low proportion of cytidine residues, multiple SD sequences and AG repeats. The latter could be identified as translation enhancer, albeit less efficient than SD sequence in several natural 5΄-UTRs.