Highly expressed proteins have an increased frequency of alanine in the second amino acid position

BACKGROUND: Although the sequence requirements for translation initiation regions have been frequently analysed, usually the highly expressed genes are not treated as a separate dataset. RESULTS: To investigate this, we analysed the mRNA regions downstream of initiation codons in nine bacteria, three archaea and three unicellular eukaryotes, comparing the dataset of highly expressed genes to the dataset of all genes. In addition to the detailed analysis of the nucleotide and codon frequencies we compared the N-termini of highly expressed proteins to the N-termini of all proteins coded in the genome. CONCLUSION: The most conserved pattern was observed at the amino acid level: strong alanine over-representation was observed at the second amino acid position of highly expressed proteins. This pattern is well conserved in all three domains of life

Translation initiation region sequence preferences in Escherichia coli

<p>Abstract</p> <p>Background</p> <p>The mRNA translation initiation region (TIR) comprises the initiator codon, Shine-Dalgarno (SD) sequence and translational enhancers. Probably the most abundant class of enhancers contains A/U-rich sequences. We have tested the influence of SD sequence length and the presence of enhancers on the efficiency of translation initiation.</p> <p>Results</p> <p>We found that during bacterial growth at 37°C, a six-nucleotide SD (AGGAGG) is more efficient than shorter or longer sequences. The A/U-rich enhancer contributes strongly to the efficiency of initiation, having the greatest stimulatory effect in the exponential growth phase of the bacteria. The SD sequences and the A/U-rich enhancer stimulate translation co-operatively: strong SDs are stimulated by the enhancer much more than weak SDs. The bacterial growth rate does not have a major influence on the TIR selection pattern. On the other hand, temperature affects the TIR preference pattern: shorter SD sequences are preferred at lower growth temperatures. We also performed an <it>in silico </it>analysis of the TIRs in all <it>E. coli </it>mRNAs. The base pairing potential of the SD sequences does not correlate with the codon adaptation index, which is used as an estimate of gene expression level.</p> <p>Conclusion</p> <p>In <it>E. coli </it>the SD selection preferences are influenced by the growth temperature and not influenced by the growth rate. The A/U rich enhancers stimulate translation considerably by acting co-operatively with the SD sequences.</p

Juhukõnnid translatsioonis

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Poomisvastus on võtmetähtsusega adaptiivsete mehhanismide regulatsioonil, mis aitavad bakteritel ebasoodsaid keskkonnatingimusi üle elada. Soolekepikeses (Escherichia coli) on selles protsessis oluliseks ensüümiks RelA, mis vastusena aminohappenäljale sünteesib signaalmolekuli (p)ppGpp. See signaalmolekul mõjutab transkriptsiooni, translatsiooni ja rakkude jagunemist. Meie töötasime välja ühe molekuli jälgimise mikroskoopia metoodika, mis võimaldab mõõta molekulide difusiooni rakus. Kasutasime seda metoodikat erineva kiirusega liikuvate molekulide kirjeldamiseks. Rakus vabalt difundeeruva valgu näiteks oli fluorestseeruv valk mEos2. Hoopis teistsuguste omadustega valguks osutus mitokondri membraanivalk Tom40, mille liikumine on ühte asukohta piiratud. RelA puhul täheldasime nii vabu, kiirelt difundeeruvaid molekule kui ka ribosoomile seondunud ja seetõttu aeglaselt liikuvaid molekule. Kombineerides ühe molekuli jälgimise tulemusi biokeemiliste andmetega, pakume välja RelA valgu töötsükli mudeli. Kuhjuv (p)ppGpp põhjustab samuti RelA aktivatsiooni. Sellisel viisil tekib positiivse tagasisidestusega regulatsioonisüsteem ja signaalmolekuli kontsentratsioon tõuseb kiiresti.The stringent response plays key role in the activation and regulation of the adaptive mechanisms that bacteria employ in order to accommodate to the adverse conditions. In E.coli the process is governed by the stringent factor RelA, transferring the amino-acid starvation signals by synthesize (p)ppGpp altering cell replication, transcription and translation. We have developed in vivo single-molecule tracking microscopy assay that allows us to track fast and slowly diffusive cytosolic (stringent factor RelA and free GFP variant mEos2) or membrane bound (mitochondrial membrane channel Tom40) proteins. The fluorescently labeled Tom40-Dendra2 complex in the mitochondrial membrane showed highly mobile but confined diffusion properties By combining biochemical and single-molecule microscopy approaches we have suggested different (p)ppGpp synthesizing mechanism from the standard hopping model where many (p)ppGpp molecules are produced upon dissociation of enzymatically active RelA from the ribosome and (p)ppGpp production is directly responsible for enhancement of the RelA enzymatic activity by positive feedback loop acting at the enzymatic level.

Codon-triplet context unveils unique features of the Candida albicans protein coding genome

<p>Abstract</p> <p>Background</p> <p>The evolutionary forces that determine the arrangement of synonymous codons within open reading frames and fine tune mRNA translation efficiency are not yet understood. In order to tackle this question we have carried out a large scale study of codon-triplet contexts in 11 fungal species to unravel associations or relationships between codons present at the ribosome A-, P- and E-sites during each decoding cycle.</p> <p>Results</p> <p>Our analysis unveiled high bias within the context of codon-triplets, in particular strong preference for triplets of identical codons. We have also identified a surprisingly large number of codon-triplet combinations that vanished from fungal ORFeomes. <it>Candida albicans </it>exacerbated these features, showed an unbalanced tRNA population for decoding its pool of codons and used near-cognate decoding for a large set of codons, suggesting that unique evolutionary forces shaped the evolution of its ORFeome.</p> <p>Conclusion</p> <p>We have developed bioinformatics tools for large-scale analysis of codon-triplet contexts. These algorithms identified codon-triplets context biases, allowed for large scale comparative codon-triplet analysis, and identified rules governing codon-triplet context. They could also detect alterations to the standard genetic code.</p

Biochemical and genetic studies of mitochondrial protein synthesis in Saccharomyces cerevisiae : characterization of the AEP3 and TRM5 gene products

textProtein synthesis in archaebacteria and the cytoplasm of eukaryotes is initiated using the initiator methionyl-tRNA (Met-tRNA[subscript i][superscript Met]). In contrast, formylated methionyltRNA (fMet-tRNA[subscript i][superscript Met][subscript f]) is found in eubacteria, and in chloroplasts and mitochondria of eukaryotes, and this formylated initiator tRNA was widely believed to be required for initiation of protein synthesis in those systems. However, the fact that initiation of protein synthesis in yeast mitochondria can occur with unformylated initiator tRNA has changed our perspective about the initiation of mitochondrial protein synthesis. This dissertation is composed of two parts. Part I describes an investigation of the yeast AEP3 gene which was isolated by a genetic screening system in Saccharomyces cerevisiae. The main goal of this part was to discover new accessory factor(s) that might be involved in initiation of protein syntheis of yeast mitochondria when there is no formylation of initiator tRNA and determine how they support the initiation process in Saccharomyces cerevisiae. The synthetic petite genetic screen identified the AEP3 gene. Protein-protein binding assays as well as protein-initiator tRNA binding assays indicate that Aep3p is associated with the initiation process in yeast mitochondrial protein synthesis. This discovery is important because it suggests the possible mechanism by which initiation of protein synthesis in yeast mitochondria occur under conditions where there is no formylation of initiator tRNA. Part II describes a study of the TRM5 gene encoding a tRNA methyltransferase in S. cerevisiae. The TRM5 gene encodes a tRNA (guanine-N1-)-methyltransferase (Trm5p) previously known to methylate guanosine at position 37 (m¹G37) in certain cytoplasmic tRNAs in S. cerevisiae. The main goal of this part was to investigate whether Trm5p is also responsible for m¹G37 modification of mitochondrial tRNAs. Full-length Trm5p, purified as a fusion protein with maltose-binding protein, exhibited robust methyltransferase activity with tRNA isolated from a [Delta]trm5 mutant strain, as well as with a synthetic mitochondrial tRNA[superscript Met][subscript f] and tRNA[superscript Phe]. High pressure liquid chromatography analysis showed the methylated product to be m¹G. Analysis of subcellular fractionation and immunoblotting revealed that the enzyme was localized to both cytoplasm and mitochondria. Our data including the analysis of N-terminal truncation mutants suggest that this tRNA modification plays an important role in reading frame maintenance in mitochondrial protein synthesis.Cellular and Molecular Biolog

Stationary phase induction of RpoS in enteric bacteria

In enteric bacteria, stress adaptation is mediated by the RpoS protein, one of several sigma-factors that, in association with RNA polymerase, collectively allow a tailored transcriptional response to environmental cues. Stress stimuli including low temperature, osmotic shock, and starvation all result in a substantial increase in RpoS abundance. Perhaps the most pronounced affect is observed during growth to stationary phase (SP) in rich medium. The mechanism of regulation depends on the specific signal, but may occur at the level of transcription, translation, protein activity or targeted proteolysis. In both Escherichia coli and Salmonella enterica cultured in rich undefined medium, the RpoS protein is barely detectable during exponential growth and increases \u3e30-fold as cells enter SP. Under these conditions, SP induction depends on transcriptional and translational control with proteolysis affecting basal levels but not regulation per se. The transiently expressed Fis protein, whose abundance inversely correlates to that of RpoS, binds just upstream of the primary rpoS promoter and represses transcription nearly 10-fold specifically during exponential growth. SP induction at the translational level relies on a novel form of genetic control dependent on the 24 nucleotides preceding the rpoS initiation codon (ribosome-binding sequence, RBS). The RNA secondary structure of the rpoS RBS is necessary and sufficient for a nearly 10-fold translational increase during SP. Control at this level is not a result of differential transcript stability, nor does it involve the known rpoS regulators ppGpp, DksA, HU, Hfq or the small regulatory RNAs, DsrA and RprA. The environmental stimuli that trigger RBS-mediated SP induction of rpoS translation also remain unknown, but similar to transcriptional control, regulation is only seen in rich undefined media. Collectively, transcriptional repression by Fis and RBS-mediated induction at the translational level account for approximately 95% of the overall SP induction of RpoS

The Role of Phage tRNAs in the Evolution of Codon Usage Biases in Giant Pseudomonas Phage phiKZ and EL

As the most abundant and diverse biological agents in the biosphere phage have significant roles in microbial ecology, acting both as lethal bacterial parasites and vehicles of horizontal gene transfer. Phage/host coevolution drives optimization of phage codon usage for use of host translational machinery, thus lowered correspondence between phage and host codon usage reduces viral fitness. Some phage may partially bypass host translational selection on their codon usage by encoding their own tRNAs, although the effects of these tRNAs on phage codon usage and translation has not been well examined. This work explores the influence of phage encoded tRNAs on viral codon usage via 1) codon usage analysis of Pseudomonas aeruginosa phage phiKZ and EL; 2) attempted engineering of phiKZ populations with deoptimized proline tRNAs; and 3) engineering and experimental evolution of mutant phiKZ strain, B1, with duplicated aspartic acid, methionine, and proline tRNAs

The Role of Initiation Factor Dynamics in Translation Initiation

Like most biological polymerization reactions, ribosome-catalyzed protein synthesis, or translation, can be divided into initiation, elongation, and termination stages. Initiation is the rate-limiting stage of translation and a critical site for translational control of gene expression. Throughout all stages of protein synthesis, the ribosome is aided by essential protein co-factors known as translation factors. I have studied the role that two translation initiation factors, IF1 and IF3, play in the mechanism and regulation of translation initiation in Escherichia coli. Specifically, I have used single-molecule fluorescence resonance energy transfer (smFRET) as a primary tool for investigating how the dynamics of IF1 and IF3 regulate the accuracy with which the translational machinery selects an initiator transfer RNA (tRNA) and the correct messenger RNA (mRNA) start codon during the initiation stage of protein synthesis

Evolution of I34 modifications in tRNAs and their role in proteome composition

[eng] Inosine is a guanosine analogue that when is found at the wobble position of the tRNAs (I34) expands its codon recognition capability. Inosine can wobble pair with cytosine, adenosine and uridine. Because inosine is not genomically encoded, essential enzymes are responsible for the hydrolytic deamination of adenosine to inosine, specifically at the wobble position of the tRNAs. In Bacteria, the modification is mostly found in tRNAArg, catalysed by the homodimeric tRNA adenosine deaminase A (TadA), with a conserved active site coordinated with an atom of Zn+2. In Eukarya, the modification is present in up to eight different tRNAs, catalysed by the heterodimeric enzyme ADAT (ADAT2-ADAT3), which originally evolved from TadA by duplication and divergence. ADAT2 is considered the catalytic subunit because it conserves the active site, whereas ADAT3, which lacks one of the essential catalytic residues, is thought to play a structural role. This substrate expansion, significantly influenced the evolution of eukaryotic genomes in terms of tRNA gene abundance and codon usage. However, the selection pressures driving this process remain unclear. In this thesis, we characterize the human transcriptome and proteome in terms of frequency and distribution of ADAT-related codons. Human codon usage indicates that I34 modified tRNAs are preferred for the translation of highly repetitive coding sequences, suggesting that I34 is an important modification for the synthesis of proteins of highly skewed amino acid composition. Persuaded by these results we extend the analysis to a series of eukaryotic and bacterial organisms, spanning the whole tree of life. We find that the preference for codons that are recognized by I34-modified tRNAs, in genes with highly biased codon composition, is universal among eukaryotes, and we report that, unexpectedly, the bacterial phylum of Firmicutes shows a similar preference. We experimentally demonstrate that the Firmicute Oenococcus oeni presents a functional expansion of I34 modification to other tRNAs other than tRNAArg, and that this process likely starts with the emergence of unmodified A34-containing tRNAs. Our findings also indicate that several ancestral bacterial groups lack both TadA and A34-tRNAs, suggesting that these species never developed the machinery to generate I34- modified tRNAs. On the other hand limited sets of bacterial species have either lost the system secondarily, or expanded it to additional tRNA substrates. In Eukaryotes, we show that a large variability in the use of I34 can be found in protists, while the modification becomes fixed in Metazoa, Fungi and Plant kingdoms.[cat] La inosina és un anàleg de la guanosina, que quan es troba a la posició 34 dels tRNAs, expandeix el nombre de codons que aquests tRNA són capaços de reconèixer. La inosina pot emparellar-se mitjançant wobbling amb citosina, adenosina i uridina. Degut que la inosina no està codificada al genoma, existeixen enzims essencials encarregats de la deaminar la adenosina a inosina específicament a la posició 34 dels tRNAs. Als organismes bacterians, aquesta modificació es troba principalment a tRNAArg i és catalitzada per l’enzim homodimeric tRNA adenosina desaminasa A (TadA), que disposa d’un centre actiu conservat. Als organismes eucariòtics, aquesta modificació és present en fins a vuit tRNAs diferents, catalitzada per l’enzim heterodimeric ADAT (ADAT2-ADAT3). Aquest enzim ha evolucionat a partir de TadA per duplicació i divergència. ADAT2 és considerat la subunitat catalítica, ja que conserva el centre actiu mentre que ADAT3 n’ha perdut un dels residus essencials i es considera que té un paper en el reconeixement dels substrats. L’expansió en el reconeixement de substrats entre TadA i ADAT ha influenciat significativament en la composició dels genomes eucariotes, particularment en l’abundància de gens de tRNA i en el biaix de la composició de codons. Tanmateix, les pressions selectives que condueixen aquests processos romanen desconegudes. En aquesta tesi, hem caracteritzat el transcriptoma i el proteoma humà respecte la freqüència i distribució de codons relacionats amb ADAT. Els nostres resultats indiquen que la composició de codons del transcriptoma humà està esbiaixada promovent una dependència en l’ús de I34, especialment en regions altament repetitives. Persuadits per aquests resultats, hem estès les nostres anàlisis a un conjunt d’organismes eucariotes i bacterians per tal de representar tot l’arbre de la vida. Hem comprovat que aquesta preferència per codons que són reconeguts per tRNAs amb I34 és generalitzada només als eucariotes, tot i que sorprenentment, també és present al fílum bacterià dels Firmicutes. Els nostres resultats també indiquen que alguns grups bacterians ancestrals no disposen de tRNAs amb A34 ni de l’enzim TadA, cosa que suggereix que aquestes espècies mai han desenvolupat la maquinària per generar tRNAs amb I34. Altres conjunts de bactèries indiquen tant la pèrdua secundària d’aquest sistema, com l’expansió a d’altres tRNAs. Hem demostrat experimentalment que Oenococcus oeni, pertanyent als Firmicutes, presenta altres tRNAs amb I34 a part del tRNAArg i que també presenta tRNA amb A34 no modificats. Entre els organismes eucariotes, els protists presenten una gran variabilitat en l’ús de tRNA amb I34, mentre que en Metazoa, Fungi i Plantae, tots els tRNAs amb I34 són presents