Olga A. Dontsova

The location of mRNA in the ribosomal 30S initiation complex; site-directed cross-linking of mRNA analogues carrying several photo-reactive labels simultaneously on either side of the AUG start codon.

Messenger RNA molecules 30–35 bases long, with sequences related to the 5′‐region of cro‐mRNA from lambda‐phage, were prepared by T7 transcription from synthetic DNA templates. Each mRNA contained five or six internal uridine residues, which were transcribed using a mixture of UTP and thio‐UTP. Initiation complexes were formed with Escherichia coli 30S ribosomes in the presence or absence of tRNA(fMet), and cross‐linking of the thio‐U residues was induced by UV irradiation at wavelengths greater than 300 nm. The cross‐linked ribosomal proteins were identified immunologically, and cross‐linked regions of the 16S RNA were isolated by excision with ribonuclease H and suitable deoxyoligonucleotides. In both cases, the particular thio‐U residue involved in the cross‐link was identified by ribonuclease T1 fingerprinting of the (radioactive) mRNA in the isolated cross‐linked complex. The principal results were that, at thio‐U positions upstream of the AUG codon, specific cross‐linking occurred to protein S7 and to the 3′‐terminus of the 16S RNA, in agreement with similar experiments using 70S ribosomes. Less specific cross‐linking was observed to proteins S1, S18 and S21 at various positions within the mRNA. Six bases downstream from the AUG codon, a tRNA‐dependent cross‐link was found to position approximately 1050 of the 16S RNA, but‐‐in contrast to similar experiments with 70S ribosomes‐‐no cross‐linking was found to the 1390–1400 region.

Telomerase: Structure, functions, and activity regulation

Telomerase is the enzyme responsible for maintenance of the length of telomeres by addition of guanine-rich repetitive sequences. Telomerase activity is exhibited in gametes and stem and tumor cells. In human somatic cells proliferation potential is strictly limited and senescence follows approximately 50–70 cell divisions. In most tumor cells, on the contrary, replication potential is unlimited. The key role in this process of the system of the telomere length maintenance with involvement of telomerase is still poorly studied. No doubt, DNA polymerase is not capable to completely copy DNA at the very ends of chromosomes; therefore, approximately 50 nucleotides are lost during each cell cycle, which results in gradual telomere length shortening. Critically short telomeres cause senescence, following crisis, and cell death. However, in tumor cells the system of telomere length maintenance is activated. Besides catalytic telomere elongation, independent telomerase functions can be also involved in cell cycle regulation. Inhibition of the telomerase catalytic function and resulting cessation of telomere length maintenance will help in restriction of tumor cell replication potential. On the other hand, formation of temporarily active enzyme via its intracellular activation or due to stimulation of expression of telomerase components will result in telomerase activation and telomere elongation that can be used for correction of degenerative changes. Data on telomerase structure and function are summarized in this review, and they are compared for evolutionarily remote organisms. Problems of telomerase activity measurement and modulation by enzyme inhibitors or activators are considered as well.

Methyltransferase That Modifies Guanine 966 of the 16 S rRNA FUNCTIONAL IDENTIFICATION AND TERTIARY STRUCTURE

N2-Methylguanine 966 is located in the loop of Escherichia coli 16 S rRNA helix 31, forming a part of the P-site tRNA-binding pocket. We found yhhF to be a gene encoding for m2G966 specific 16 S rRNA methyltransferase. Disruption of the yhhF gene by kanamycin resistance marker leads to a loss of modification at G966. The modification could be rescued by expression of recombinant protein from the plasmid carrying the yhhF gene. Moreover, purified m2G966 methyltransferase, in the presence of S-adenosylomethionine (AdoMet), is able to methylate 30 S ribosomal subunits that were purified from yhhF knock-out strain in vitro. The methylation is specific for G966 base of the 16 S rRNA. The m2G966 methyltransferase was crystallized, and its structure has been determined and refined to 2.05Å. The structure closely resembles RsmC rRNA methyltransferase, specific for m2G1207 of the 16 S rRNA. Structural comparisons and analysis of the enzyme active site suggest modes for binding AdoMet and rRNA to m2G966 methyltransferase. Based on the experimental data and current nomenclature the protein expressed from the yhhF gene was renamed to RsmD. A model for interaction of RsmD with ribosome has been proposed.

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.

Identification of Escherichia coli m2G methyltransferases: II. The ygjO gene encodes a methyltransferase specific for G1835 of the 23 S rRNA.

Escherichia coli ribosomal RNA contains five guanosine residues methylated at N2. The ygjO gene was previously predicted to methylate 16 S rRNA residue G966 due to its high sequence homology with the protein RsmC, responsible for G1207 methylation. We have identified the target of YgjO as being m2G1835 of the 23 S rRNA and not m2G966 of the 16 S rRNA as expected. Knock-out of the ygjO gene leads to loss of modification at G1835, as revealed by reverse transcription. Moreover, the modification could be restored by in vivo complementation of the ygjO knock-out strain with a plasmid expressing ygjO. Recombinant YgjO protein is able to methylate in vitro protein-free 23 S rRNA, but not assembled 50 S subunits purified from the ygjO knock-out strain. The nucleotide m2G1835 is located in a functionally extremely important region of the ribosome, being located within intersubunit bridges of group B2. Growth competition assays reveal that the lack of the G1835 methylation causes growth retardation, especially at temperatures higher than optimal and in poor media. Based on these results we suggest that YgjO be renamed to RlmG in accordance with the accepted nomenclature for rRNA methyltransferases.

Genome sequence and analysis of methylotrophic yeast Hansenula polymorpha DL1

BackgroundHansenula polymorpha DL1 is a methylotrophic yeast, widely used in fundamental studies of methanol metabolism, peroxisome biogenesis and function, and also as a microbial cell factory for production of recombinant proteins and metabolic engineering towards the goal of high temperature ethanol production.ResultsWe have sequenced the 9 Mbp H. polymorpha DL1 genome and performed whole-genome analysis for the H. polymorpha transcriptome obtained from both methanol- and glucose-grown cells. RNA-seq analysis revealed the complex and dynamic character of the H. polymorpha transcriptome under the two studied conditions, identified abundant and highly unregulated expression of 40% of the genome in methanol grown cells, and revealed alternative splicing events. We have identified subtelomerically biased protein families in H. polymorpha, clusters of LTR elements at G + C-poor chromosomal loci in the middle of each of the seven H. polymorpha chromosomes, and established the evolutionary position of H. polymorpha DL1 within a separate yeast clade together with the methylotrophic yeast Pichia pastoris and the non-methylotrophic yeast Dekkera bruxellensis. Intergenome comparisons uncovered extensive gene order reshuffling between the three yeast genomes. Phylogenetic analyses enabled us to reveal patterns of evolution of methylotrophy in yeasts and filamentous fungi.ConclusionsOur results open new opportunities for in-depth understanding of many aspects of H. polymorpha life cycle, physiology and metabolism as well as genome evolution in methylotrophic yeasts and may lead to novel improvements toward the application of H. polymorpha DL-1 as a microbial cell factory.

Function of the ribosomal E-site: a mutagenesis study

Ribosomes synthesize proteins according to the information encoded in mRNA. During this process, both the incoming amino acid and the nascent peptide are bound to tRNA molecules. Three binding sites for tRNA in the ribosome are known: the A-site for aminoacyl-tRNA, the P-site for peptidyl-tRNA and the E-site for the deacylated tRNA leaving the ribosome. Here, we present a study of Escherichia coli ribosomes with the E-site binding destabilized by mutation C2394G of the 23S rRNA. Expression of the mutant 23S rRNA in vivo caused increased frameshifting and stop codon readthrough. The progression of these ribosomes through the ribosomal elongation cycle in vitro reveals ejection of deacylated tRNA during the translocation step or shortly after. E-site compromised ribosomes can undergo translocation, although in some cases it is less efficient and results in a frameshift. The mutation affects formation of the P/E hybrid site and leads to a loss of stimulation of the multiple turnover GTPase activity of EF-G by deacylated tRNA bound to the ribosome.

Ribosomal RNA guanine-(N2)-methyltransferases and their targets.

Five nearly universal methylated guanine-(N2) residues are present in bacterial rRNA in the ribosome. To date four out of five ribosomal RNA guanine-(N2)-methyltransferases are described. RsmC(YjjT) methylates G1207 of the 16S rRNA. RlmG(YgjO) and RlmL(YcbY) are responsible for the 23S rRNA m2G1835 and m2G2445 formation, correspondingly. RsmD(YhhF) is necessary for methylation of G966 residue of 16S rRNA. Structure of Escherichia coli RsmD(YhhF) methyltransferase and the structure of the Methanococcus jannaschii RsmC ortholog were determined. All ribosomal guanine-(N2)-methyltransferases have similar AdoMet-binding sites. In relation to the ribosomal substrate recognition, two enzymes that recognize assembled subunits are relatively small single domain proteins and two enzymes that recognize naked rRNA are larger proteins containing separate methyltransferase- and RNA-binding domains. The model for recognition of specific target nucleotide is proposed. The hypothetical role of the m2G residues in rRNA is discussed.

Structural and functional analysis of 5S rRNA in Saccharomyces cerevisiae

Abstract5S rRNA extends from the central protuberance of the large ribosomal subunit, through the A-site finger, and down to the GTPase-associated center. Here, we present a structure-function analysis of seven 5S rRNA alleles which are sufficient for viability in the yeast Saccharomyces cerevisiae when expressed in the absence of wild-type 5S rRNAs, and extend this analysis using a large bank of mutant alleles that show semi-dominant phenotypes in the presence of wild-type 5S rRNA. This analysis supports the hypothesis that 5S rRNA serves to link together several different functional centers of the ribosome. Data are also presented which suggest that in eukaryotic genomes selection has favored the maintenance of multiple alleles of 5S rRNA, and that these may provide cells with a mechanism to post-transcriptionally regulate gene expression.

How can elongation factors EF-G and EF-Tu discriminate the functional state of the ribosome using the same binding site?

Elongation factors EF‐G and EF‐Tu are structural homologues and share near‐identical binding sites on the ribosome, which encompass the GTPase‐associated centre (GAC) and the sarcin‐ricin loop (SRL). The SRL is fixed structure in the ribosome and contacts elongation factors in the vicinity of their GTP‐binding site. In contrast, the GAC is mobile and we hypothesize that it interacts with the alpha helix D of the EF‐Tu G‐domain in the same way as with the alpha helix A of the G′‐domain of EF‐G. The mutual locations of these helices and GTP‐binding sites in the structures of EF‐Tu and EF‐G are different. Thus, the orientation of the GAC relative to the SRL determines whether EF‐G or EF‐Tu will bind to the ribosome.