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>Magister Scientiae - MSc / Heterologous metagenomic screening strategies have relied largely on the construction of DNA libraries and screening in Escherichia coli to access novel enzymes. There is an increased demand for the identification of novel lignocellulose degrading enzymes with enhanced biochemical properties which are suitable for applications in industrial processes; biofuels being one of them. The use of heterologous gene expression in function based metagenomic studies has resulted in the discovery of enormous novel bioactive compounds. However, there are limitations associated with using E. coli as a heterologous host which does not allow transcription and translation of all genes in the metagenome. E. coli can only express 40% of the environmental DNA because of promoter recognition, codon usage, and host toxicity of gene products. Therefore alternative strategies for expressing or producing novel enzymes are needed, which can also be employed in metagenomic gene discovery. In vitro protein synthesis is an important tool in molecular biology and used to obtain proteins from genes for functional and expression studies. These systems may hold the key to unlock more of the potential in metagenomic DNA. The broader aim of the study is to develop non- E. coli based cell-free protein synthesis systems to further the metagenomics screening. In this study, Rhodococcus erythropolis H8 was evaluated for its suitability in cell-free expression. Crude extracts containing the macromolecular components (70S or 80S ribosomes, tRNAs, initiation, elongation and termination factors) fromR. erythropolis were prepared using existing crude extract based cell-free protein synthesis (CFPS) protocols. Three genes were selected and used as templates for synthesis: cell11, xp12 and acetyl xylan esterase (axe10), all previously isolated from metagenomic libraries screened inE. coli. As judged by zymograms and enzyme assays, all enzymes were successfully expressedfrom their native promoters and in recombinants clones using the PtipA promoter, and wereactive. Furthermore, the amounts of XP12 protein produced using pFos-XP_12 was 1.2mg/mlfrom E. coli and 1.67mg/ml from R. erythropolis CFPS, showing that the R. erythropolismachinery was more efficient in the expression of XP12 than the E. coli machinery. To the best of our knowledge this is the first demonstration of a cell-free expression using an actinomycete.

Ribosomal incorporation of unnatural amino acids (AAs) into peptides or proteins has found broad applications in studying translation mechanism, discovering potential therapeutics, and probing protein structure and function. However, such applications are generally limited by the low incorporation efficiencies of the unnatural AAs. With in vitro kinetics studies using a purified E. coli translation system, we found that the natural N-alkyl AA carrier, tRNAPro, could hasten the incorporation of N-methyl AAs. Also, the incorporation rate increased remarkably with increasing pH in the range of 7 to 8.5, suggesting the rate was limited by peptidyl transfer, not accommodation. Competition experiments revealed that several futile cycles of delivery and rejection of the A site N-methyl AA-tRNA were required per peptide bond formation, and the incorporation yield could be increased by using a higher Mg2+ concentration. Kinetics of ribosomal polymerization, using AA-tRNA substrates prepared from the standard N-NVOC-AA-pdCpA chemoenzymatic ligation method, clarified that the inefficiency of incorporation was due to the penultimate dC. This dC prompted faster peptidyl-tRNA drop-off, leading to loss of processivities along consecutive incorporations. Circumventing the penultimate dC by using our N-NVOC-AA-pCpA chemoenzymatic ligation or the flexizyme charging method to prepare the AA-tRNA substrates was able to improve the efficiencies of ribosomal consecutive incorporations of unnatural AAs. By studying the translation steps after aminoacylation of tRNAPyl, the favored carrier for unnatural AAs in vivo, we demonstrated surprisingly slow biphasic kinetics of tRNAPyl-mediated amber suppression in vitro. The fast phase amplitude increased with increasing EF-Tu concentration, allowing measurement of Kd of EF-Tu binding. Results revealed ~25-fold weaker EF-Tu binding affinity of the tRNAPyl body than that of E. coli tRNAPhe. The fast phase rate was ~30-fold slower than that of native substrates, and this rate was limited by the ~10-fold less efficient AA-tRNAPyl:EF-Tu:GTP ternary complex binding to the ribosome. The incorporation was so slow that termination by RF2 mis-reading of the amber codon became a significant competing reaction. The processivity was unexpectedly impaired as ~40% of the dipeptidyl-tRNAPyl could not be elongated to tripeptide. This new overall understanding opens a window of improving unnatural AA incorporation both in vitro and in vivo.

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Title: The Relationship between Moderate, Within Day Protein Intake and Energy Balance on Body Composition of Collegiate Sand Volleyball Players Background: Achieving an ideal body composition with relatively low fat mass and relatively high fat-free mass (FFM) is desirable for virtually all competitive athletes. Some studies suggest that protein intake, depending on quality, amount, and timing, may improve relative musculature by stimulating muscle protein synthesis, but some issues related to timing and amount of protein intake remain unclear. Current evidence suggests that frequent consumption of moderate amounts of protein is useful for muscle building. Purpose: The purpose of this study was to simultaneously assess energy balance and protein intake to determine if these factors are associated with body composition in a population of collegiate sand volleyball players. Methods: In a cross sectional, observational study, players completed a food intake and activity form for a 24-hour period to serve as the basis of energy balance and protein intake assessment. The assessment day was representative of a typical day during the regular training season. These data were entered into a software program providing total and hourly energy balance and nutrient content of the consumed foods. Athletes were measured for body composition via a multi-current bioelectrical impedance scale to predict weight, BMI, fat mass and fat free mass. Height was measured using a standard wall-mounted stadiometer. Data analyses included descriptive and frequency statistics, Spearman correlations and regression analyses. Results: Twelve women from the GSU sand volleyball team participated in the study using an IRB-approved protocol. The mean BMI was 22 kg/m2 (±3 kg/m2) and the mean body fat percentage was 18% (±7%). The mean protein intake for all participants was 132 grams (±52 g). Protein intake distribution was skewed, on average, toward the latter half of the day with approximately 19% of protein consumed in the morning and 34% consumed in the evening. The mean net energy balance at the end of the 24-hour assessment period was -404 (±385) kcal. Athletes, on average, spent 17 hours in a catabolic energy balance state (< 0 kcal). No significant correlation was found between energy balance per gram of protein consumption and body composition. However, regression analyses indicated that energy balance and protein variables explain a significant proportion (p=.037) of the variance in body fat percentage. Conclusions: Sand volleyball players in this study spent a high proportion of time in a negative energy balance, which may have compromised the potential benefit that frequent protein consumption may have had on FFM. Since both energy balance and protein explain a significant proportion of the variance in body composition, these athletes might benefit from improving within-day energy balance as a strategy for optimizing body composition.

In the only human study to date arginine supplementation stimulated tumour protein synthesis in patients with breast cancer. However, it is not known if these stimulatory effects of arginine are limited to breast cancer, and the mechanisms of its action are not well understood. Moreover it would be important to find out whether the action of arginine is selective for malignant tissues or if it can affect normal, healthy tissues. The first study investigated the effect of arginine supplements for 3 days on nitrogen balance and on the liver synthesis rate of albumin in healthy volunteers. The addition of arginine to the diet produced a remarkable nitrogen retention over the three days. No changes in the albumin synthesis rates were detected between the two dietary periods. No effects were observed on the protein synthesis, RNA content or transcriptional efficiency in a variety of the tissues when adult rats were supplemented with arginine for three days. In vitro studies using a human breast tumour cell line (MCF-7) showed that arginine is an essential nutrient for the proliferation of breast tumour cells. Neither the direct precursor for polyamine synthesis, ornithine, nor the polyamine putrescine could replace arginine for growth. Only citrulline could completely substitute arginine for protein synthesis and growth. The arginine:nitric oxide pathways did not seem involved in the stimulatory effects of arginine. The main reason for the high arginine requirement was found to be the production of the enzyme arginase. The final study investigated the in vivo effects of arginine supplements on human tumours. Arginine supplementation did not affect the tumour protein synthesis rates in patients with head and neck tumours, suggesting that not all human tumours are stimulated by arginine.

Metabolism of the transport protein albumin is known to be regulated by long-term nutrient status. To assess the short-term response to feeding, studies were performed which measured the rate of albumin synthesis once after an overnight fast and once after one of two feeding regimens consisting of five small hourly meals or one large meal. Albumin synthesis increased by approximately 25% and 31% above the fasting value for the small meals and large meal regimens respectively, thus demonstrating that albumin synthesis is acutely sensitive to nutrient intake. A supplementary animal experiment indicated that this response is part of an overall increase in liver protein synthesis. Elevated plasma fibrinogen concentration has been implicated as a risk factor for the development of cardiovascular disease. As cigarette smokers are known to have a significantly raised plasma fibrinogen concentration, studies were performed to investigate the metabolic mechanism responsible for this by comparing the rate of fibrinogen synthesis in smokers and non-smokers and smokers who had abstained from smoking for 14 days. Smokers had a significantly greater absolute rate of fibrinogen synthesis (mean SD, 21.5 1.9 versus 16.0 1.4 mg/kg/d, p<0.05). Although smokers were found to have moderate leucocytosis, there did not appear to be any increased metabolic activity of lymphocytes. In a separate group of smokers, abstention from smoking for 14 days resulted in a significant reduction in plasma fibrinogen concentration of 19%. The fractional rate of fibrinogen synthesis also decreased significantly by 14% suggesting that a substantial part of the hyperfibrinogenaemia observed in smokers is the product of increased output by the liver.

Termination of protein synthesis occurs in response to the translocation of a stop codon (UAA, UAG or UGA) into the A site of the ribosome. Unlike sense codons, stop signals in the mRNA are recognized by two classes of specialized proteins called release factors (RFs): the class I or decoding RF, which recognizes the stop codon and promotes peptidyl-tRNA hydrolysis and class II RF, a G-protein that promotes the dissociation of the decoding RF from the ribosome. The discovery that stop codons are decoded by a protein factor rather than a specific tRNA opened up the possibility that the signal for termination of protein synthesis might extend beyond the stop codon itself. Biochemical and genetic experiments in prokaryotes confirmed that bias in nucleotide usage around stop codons correlates with translation termination efficiency. The objective of the current investigation was to define the eukaryotic termination signal by determining the bias in the nucleotide sequence surrounding eukaryotic stop codons and to identify whether this was a determinant of translation termination efficiency. Bioinformatic analysis of five diverse eukaryotic genomes was undertaken to identify potential eukaryotic translation termination signal elements. Significant nucleotide bias was identified both 5� and 3� of the stop codon in all the genomes investigated. Correlations were identified between nucleotide bias and gene expression levels, and between nucleotide bias and natural recoding sites predicting that nucleotides 5� and 3� of the stop codon affect termination efficiency. These correlations were common to all organisms investigated and suggested the existence of a eukaryotic termination signal. Termination signals identified from the bioinformatic analysis were assayed to determine the efficiency of termination in an in vitro dual luciferase reporter assay. Results indicated that nucleotides both 5� and 3� of the stop codon could significantly alter termination signal efficiency, although readthrough did not vary by greater than 1%. The effect of nucleotides 3� to the stop codon on termination efficiency was investigated further in mammalian cultured cells using the dual luciferase reporter assay. Results showed a significant relationship between the identity of these nucleotides and observed termination efficiencies with nucleotides at positions +4 and +8 giving the strongest correlation. Termination sequence elements of the form UGA CUN NCN mediated up to 5% readthrough in cultured cells. Investigations into the underlying mechanisms that were responsible for the variation in termination efficiency were also undertaken. Co-transfection of specific suppressor tRNAs enhanced but did not change the pattern of observed termination efficiency, indicating that the mechanisms mediated by the termination signal element was not mediated through suppressor tRNA binding. Alignments of 18S rRNA sequences indicated potential extensive interactions between the rRNA and the mRNA termination signal element. Experiments that assessed the effect of eRF1 levels on termination at inefficient termination signals in vitro revealed that increased levels of eRF1 could improve termination efficiency. These results indicate that, as in prokaryotes, specific nucleotides beyond the stop codon modulate translation termination efficiency in eukaryotes, and that the translation termination signal should be considered a sequence element.

The aminoacyl-tRNA synthetases are among the major protein components in the translation machinery. These essential proteins are responsible for charging their cognate tRNAs with the correct amino acid. Mammalian arginyl-tRNA synthetase (ArgRS), unlike all other eukaryotic aminoacyl-tRNA synthetases, is unique due to the coexistence of two structurally distinct forms of the same enzyme within the same cell: a complexed (or high molecular weight) form that is part of the multi-synthetase complex, and a free (or low molecular weight) form. Until now, not much information is known as to why the cell would synthesize and utilize two different forms of the same enzyme. Do the two forms of ArgRS perform similar or different biological functions? The main hypothesis that was originally proposed is that only the complexed form of ArgRS plays a crucial role in protein synthesis, while the free form of this enzyme participates in the ubiquitination pathway by tagging proteins with acidic NH2-termini (destined for degradation) with an arginine residue on their NH2-terminal end which will serve as a signal for ubiquitin-mediated destruction. Based on my studies, the data indicate that the high molecular weight form of ArgRS, which is present exclusively as an integral component of the multisynthetase complex, is essential for normal protein synthesis and growth of CHO cells even when low molecular weight, free ArgRS is present and Arg-tRNA continues to be synthesized at close to wild type levels. Based on these observations, we can conclude that Arg-tRNA generated by the synthetase complex is a more efficient precursor for protein synthesis than Arg-tRNA generated by free ArgRS, exactly as would be predicted by the channeling model for mammalian translation. No phenotype has been determined for cells expressing only the complexed form of ArgRS, and no direct interaction has been observed between ArgRS and arginyl-tRNA-protein transferase (ATE). Based on this information, we suggest that the function(s) of the free form of ArgRS is either not necessary or is performed by the complexed form when the free form is missing.