Studies have discovered higher frequencies of single nucleotide polymorphisms (SNPs) in different mitochondrial genes are associated with subnormozoospermia. However, the frequencies of SNPs in ND1 and ND2 are not unknown. The present research was aimed to determine the frequencies of SNPs in ND1 and ND2 genes of the mitochondrial genome in fertile and subfertile men and whether changes in codon usage was associated with fertility phenotypes. Total genomic DNA from 157 semen samples was extracted using the proteinase K/SDS digestion procedure, followed by phenol/chloroform purification and ethanol precipitation. ND1 and ND2 genes were amplified respectively from 80 and 92 DNA samples from different fertility groups. Each PCR product was sequenced to identify mutations. Codon change resulting from a nucleotide substitution was determined by comparison with a reference mtDNA sequence obtained from the NCBI database. The frequency of codon usage in the reference mtDNA was determined by the computer program MEGA version 2.1. Eleven synonymous nucleotide substitutions and two non-synonymous substitutions were found in this study. Four SNPs were previously characterized; all SNPs were homoplasmic. None of the SNPs were likely to affect the function of the proteins on the basis of the hydrophobicity plots or secondary structure predictions. Sixty two percent of synonymous mutations were found to change from a high to a low relative codon usage values; 37% of synonymous mutations changed from a low to a high relative usage value. Chi-square (χ²) test (χ²= 0.067 with 1 d.f.) showed that there was no significant difference at the 5% level between these changes. Thus, change in codon usage was not related to semen quality in men. Further, there were no statistically significant differences in the observed frequencies of SNPs of fertile and subfertile men. However, the sample size was small and this study was only focused on a single NZ Caucasian population. Further study including larger and more diverse population samples may provide further insight into the functional importance of codon usage and its relevance to fertility

Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.

Thesis advisor: Abhishek Chatterjee / Thesis advisor: Eranthie Weerapana / In nature, just twenty canonical amino acids are responsible for the creation of nearly all proteins. Genetic code expansion (GCE), or the incorporation of noncanonical amino acids (ncAAs) into living cells, is a powerful tool that expands the studies we are capable of performing using proteins. This technology relies on engineered aminoacyl-tRNA synthetase (aaRS)/tRNA pairs that are orthogonal to the host cells’ endogenous aaRS/tRNA pairs, and one of the main limitations of GCE arises from the inefficiency of these suppressor tRNAs when expressed in a foreign host cell. To address this limitation, we have previously reported a strategy for the virus-assisted directed evolution of tRNAs (VADER) which is uniquely capable of addressing the specific needs of tRNA evolution. In order to advance the capabilities of VADER, we made a number of modifications to the VADER selection scheme. First, we designed and executed a modified VADER selection that enabled the evolution of a new class of tRNAs, and with this VADER selection, we were able to generate a first-generation E. coli tyrosyl tRNA (tRNATyr) variant that was three times as active as its wild-type equivalent. Next, we introduced a number of refinements to the VADER strategy to generate VADER 2.0, an improved workflow capable of screening larger libraries and libraries encoding more active variants. Using VADER 2.0, we created second-generation tRNAPyl and tRNATyr mutants that achieved incorporation efficiencies that were greater than five-fold higher than their wild-type equivalents across a wide variety of substrates, enabling exciting GCE experiments that would not be possible otherwise. / Thesis (PhD) — Boston College, 2023. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

The human immunodeficiency virus type 1 (HIV-1) uses a mechanism of genetic recoding known as programmed ribosomal frameshifting to translate the proteins encoded by the pol gene. The pol gene overlaps the preceding gag gene in the -1 reading frame relative to gag. It contains neither a start codon nor an internal ribosome entry site (IRES) to initiate translation of its proteins. Rather the host ribosomes are forced to pause due to tension placed on the mRNA when they encounter a specific secondary structural element in the mRNA. This tension is relieved by disruption of the contacts between the mRNA codons and tRNA anticodons at a �slippery� sequence within the ribosomal decoding centre. Re-pairing of the tRNAs occurs in the new -1 frame after movement of the mRNA backwards by one nucleotide, allowing the ribosome to translate the pol gene as a Gag-Pol polyprotein. A change in ratio of Gag to Gag-Pol proteins affects viral assembly, and most significantly dramatically reduces viral infectivity. The prevailing model for the mechanism of -1 frameshifting has focussed on a pre-translocational event, where slippage occurs when the slippery sequence is within the ribosomal A and P sites. This model precludes a contribution from the codon immediately downstream of the slippery sequence leading into the secondary structural element. I have termed this the �intercodon�. Often at frameshifting sites it is a termination codon, whereas in HIV-1 it is a glycine codon, GGG. When the intercodon within the frameshift element was changed from the wild-type GGG to a termination codon UGA, the efficiency of frameshifting decreased 3-4-fold in an in vivo assay in cultured human cells. This result mimicked previous data in the group within bacterial cells and cultured monkey COS-7 cells. Changing the first nucleotide of the intercodon to each of the three other bases altered frameshifting to varying degrees, but not following expected patterns for base stacking effects. Such a result would support a post-translocational model for -1 frameshifting. It suggested that the intercodon might be within the ribosomal A site before frameshifting, and that the slippery sequence was therefore within the P and E sites. This was investigated by modulating the expression of decoding factors for the intercodon - the release factor eRF1 and cognate suppressor tRNAs when it was either of the UGA or UAG termination codons, and tRNA[Gly] for the native GGG glycine codon. These were predicted to affect frameshifting only if slippage were occurring when the ribosomal elongation cycle was in the post-translocational state. Overexpression of tRNA[Gly] gave inconsistent effects on frameshifting in vivo, implying that its concentration may not be limiting within the cell. When eRF1 was overexpressed or depleted by RNAi, significant functional effects of decreased or increased stop codon readthrough respectively were documented. Expression of suppressor tRNAs increased readthrough markedly in a stop codon-specific manner. These altered levels of eRF1 expression were able to modulate the +1 frameshifting efficiency of the human antizyme gene. Overexpression of eRF1 caused significant reduction of frameshifting of the HIV-1 element with the UAG or UGA intercodon. Depletion of the protein by contrast had unexplained global effects on HIV-1 frameshifting. Suppressor tRNAs increased frameshifting efficiency at the UAG or UGA specifically in a cognate manner. These results strongly indicate that a post-translocational mechanism of frameshifting is used to translate the HIV-1 Gag-Pol protein. A new model (�almost� post-translocational) has been proposed with -1 frameshifting occurring for 1 in 10 or 20 ribosomal passages during the end stages of translocation, because of opposing forces generated by translocation and by resistance to unwinding of the secondary structural element. With translocation still incomplete the slippery sequence is partially within the E and P sites, and the intercodon partially within the A site. The nature of the intercodon influences frameshifting efficiency because of how effectively the particular decoding factor is able to bind to the partially translocated intercodon and maintain the normal reading frame.

Over the last twenty years, the expansion of the genetic code has been made possible by the encoding of unnatural amino acids into proteins. Unnatural amino acids could be used to expand the chemical functionalities available to biology allowing for the production of ‘allo-proteins’ with potentially novel structures and functions. One method to engineer the genetic code is to engineer the translational components responsible for its maintenance. This methodology relies primarily on the evolution of the aminoacyl tRNA synthetases and their cognate tRNAs to produce an orthogonal enzyme and tRNA pair that allows for the insertion of unnatural amino acids into proteins. To date only a handful of these orthogonal pairs are available for use in genetic code expansion. As in vitro and in vivo techniques to re-code the genetic code have expanded, the utility of having multiple orthogonal pairs to site-specifically insert multiple unnatural amino acids into proteins has increased. In addition, the development of a variety of orthogonal pairs based on the twenty canonical aminoacyl tRNA synthetase-tRNA pairs will expand the types of unnatural amino acid sidechains available for protein engineering efforts. Herein we describe the engineering of the tryptophanyl tRNA synthetase and tRNA superscript Trp], pair from yeast for use as an orthogonal pair in E. coli. We have successfully built and tested synthetic expression constructs for the expression of this orthogonal pair in vivo. In addition, we have rationally engineered an orthogonal amber nonsense suppressor tRNA based on the yeast tRNA[superscript Trp], dubbed AS3.4. This suppressor has been shown to be an efficient orthogonal suppressor tRNA in vivo, and will aid in our efforts to expand the genetic code with heterocyclic unnatural amino acids. We also have developed a potentially tunable two part selection scheme, for use in the directed evolution of mutant tRNA synthetases that are specific to unnatural amino acid substrates. / text

In unstimulated cells, the transcription factor NF-κB is held in the cytoplasm in an inactive state by IκB inhibitor proteins. Activation of NF--KB is mediated by signal induced degradation of IκBα via the ubiquitin proteasome-dependent pathway. Targeting the proteins for ubiquitin-mediated proteolysis is an irrevocable decision, and as such, the process needs to be highly specific and tightly regulated. This task is achieved by conjugation and deconjugation enzymes that act in a dynamic and coordinated mechanism. In a yeast two hybrid screen designed to identify proteins involved in IκBα signalling Ubch9 was found to interact with the N-terminal regulatory region of IκBα. Although Ubch9 is an enzyme homologous to E2 ubiquitin conjugating enzymes we have shown that is unable to form a thioester with ubiquitin but it is capable to form a thioester with the small ubiquitin-like protein SUMO- 1. To fully characterise the SUMO-1 modification reaction we have purified the proteins and cloned the genes encoding the SUMO-1 activating enzyme (SAEl/SAE2) and shown that it is homologous to enzymes involved in the activation of ubiquitin, Smt3p, the yeast SUMO-1 homologue, and Rublp/Nedd8, another ubiquitin-like protein. SUMO-1 is conjugated to target proteins by a pathway that is distinct from, but analogous to, ubiquitin conjugation. SUMO-1 was efficiently conjugated, both in vivo and in vitro, to IκBα on lysine 21, which is also utilised for ubiquitin modification. Thus, by blocking ubiquitination SUMO-1 modification acts antagonistically to generate a pool of IκBα resistant to proteasome-mediated degradation which consequently inhibits NF-κB dependent transcription activation. In view of several lines of similarity between NF-kB and p53, the involvement of SUMO-1 modification in the metabolism of the tumour supressor p53 was investigated. We have shown that p53 is modified by SUMO-1 at a single site, lysine 386 in the C-terminus of p53. Although p53 is regulated by ubiquitination, SUMO-1 and ubiquitin modification do not compete for the same lysine in p53. However, overexpression of SUMO-1 activates the transcriptional activity of wild type p53, but not K386R p53 where the SUMO-1 acceptor site has been mutated. A consensus sequence was obtained by comparison of the sequences surrounding the SUMO-1 acceptor lysine in proteins that have been shown to be modified by SUMO-1 and revealed a possible recognition site for SUMO-1 conjugation machinery. Tagging of proteins with SUMO-1 regulates transcriptional activation, either by interfering with subcellular location or with the ubiquitination pathway. The pathway may represent a novel target for drug development.

The human immunodeficiency virus type 1 (HIV-1) uses a mechanism of genetic recoding known as programmed ribosomal frameshifting to translate the proteins encoded by the pol gene. The pol gene overlaps the preceding gag gene in the -1 reading frame relative to gag. It contains neither a start codon nor an internal ribosome entry site (IRES) to initiate translation of its proteins. Rather the host ribosomes are forced to pause due to tension placed on the mRNA when they encounter a specific secondary structural element in the mRNA. This tension is relieved by disruption of the contacts between the mRNA codons and tRNA anticodons at a �slippery� sequence within the ribosomal decoding centre. Re-pairing of the tRNAs occurs in the new -1 frame after movement of the mRNA backwards by one nucleotide, allowing the ribosome to translate the pol gene as a Gag-Pol polyprotein. A change in ratio of Gag to Gag-Pol proteins affects viral assembly, and most significantly dramatically reduces viral infectivity. The prevailing model for the mechanism of -1 frameshifting has focussed on a pre-translocational event, where slippage occurs when the slippery sequence is within the ribosomal A and P sites. This model precludes a contribution from the codon immediately downstream of the slippery sequence leading into the secondary structural element. I have termed this the �intercodon�. Often at frameshifting sites it is a termination codon, whereas in HIV-1 it is a glycine codon, GGG. When the intercodon within the frameshift element was changed from the wild-type GGG to a termination codon UGA, the efficiency of frameshifting decreased 3-4-fold in an in vivo assay in cultured human cells. This result mimicked previous data in the group within bacterial cells and cultured monkey COS-7 cells. Changing the first nucleotide of the intercodon to each of the three other bases altered frameshifting to varying degrees, but not following expected patterns for base stacking effects. Such a result would support a post-translocational model for -1 frameshifting. It suggested that the intercodon might be within the ribosomal A site before frameshifting, and that the slippery sequence was therefore within the P and E sites. This was investigated by modulating the expression of decoding factors for the intercodon - the release factor eRF1 and cognate suppressor tRNAs when it was either of the UGA or UAG termination codons, and tRNA[Gly] for the native GGG glycine codon. These were predicted to affect frameshifting only if slippage were occurring when the ribosomal elongation cycle was in the post-translocational state. Overexpression of tRNA[Gly] gave inconsistent effects on frameshifting in vivo, implying that its concentration may not be limiting within the cell. When eRF1 was overexpressed or depleted by RNAi, significant functional effects of decreased or increased stop codon readthrough respectively were documented. Expression of suppressor tRNAs increased readthrough markedly in a stop codon-specific manner. These altered levels of eRF1 expression were able to modulate the +1 frameshifting efficiency of the human antizyme gene. Overexpression of eRF1 caused significant reduction of frameshifting of the HIV-1 element with the UAG or UGA intercodon. Depletion of the protein by contrast had unexplained global effects on HIV-1 frameshifting. Suppressor tRNAs increased frameshifting efficiency at the UAG or UGA specifically in a cognate manner. These results strongly indicate that a post-translocational mechanism of frameshifting is used to translate the HIV-1 Gag-Pol protein. A new model (�almost� post-translocational) has been proposed with -1 frameshifting occurring for 1 in 10 or 20 ribosomal passages during the end stages of translocation, because of opposing forces generated by translocation and by resistance to unwinding of the secondary structural element. With translocation still incomplete the slippery sequence is partially within the E and P sites, and the intercodon partially within the A site. The nature of the intercodon influences frameshifting efficiency because of how effectively the particular decoding factor is able to bind to the partially translocated intercodon and maintain the normal reading frame.

Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2007. / Includes bibliographical references (leaves 115-120). Also available in electronic version.

Reading of messenger RNA (mRNA) by aminoacyl-tRNAs (aa-tRNAs) on the ribosomes in the bacterial cell occurs with high accuracy. It follows from the physical chemistry of enzymatic reactions that there must be a trade-off between rate and accuracy of initial tRNA selection in protein synthesis: when the current accuracy, the A-value, approaches its maximal possible value, the d-value, the kinetic efficiency of the reaction approaches zero. We have used an in vitro system for mRNA translation with purified E. coli components to estimate the d- and A-values by which aa-tRNAs discriminate between their cognate and near cognate codons displayed in the ribosomal A site. In the case of tRNALys, we verified the prediction of a linear trade-off between kinetic efficiency of cognate codon reading and the accuracy of codon selection. These experiments have been extended to a larger set of tRNAs, including tRNAPhe, tRNAGlu, tRNAHis, tRNACys, tRNAAsp and tRNATyr, and linear efficiency-accuracy trade-off was observed in all cases. Similar to tRNALys, tRNAPhe discriminated with higher accuracy against a particular mismatch in the second than in the first codon position. Remarkably high d-values were observed for tRNAGlu discrimination against a C-C mismatch in the first codon position (70 000) and for tRNAPhe discrimination against an A-G mismatch in the second codon position (79 000). At the same time, we have found a remarkably small d-value (200) for tRNAGlu misreading G in the middle position of the codon (U-G mismatch). Aminoglycoside antibiotics induce large codon reading errors by tRNAs. We have studied the mechanism of aminoglycoside action and found that the drug stabilized aminoacyl-tRNA in a codon selective in relation to a codon non-selective state. This greatly enhanced the probability of near cognate aminoacyl-tRNAs to successfully transcend the initial selection step of the translating ribosome. We showed that Mg2+ ions, in contrast, favour codon non-selective states and thus induce errors in a principally different way than aminoglycosides.  We also designed experiments to estimate the overall accuracy of peptide bond formation with, including initial selection accuracy and proofreading of tRNAs after GTP hydrolysis on EF-Tu. Our experiments have now made it possible to calibrate the accuracy of tRNA selection in the test tube to that in the living cells. We will now also be able to investigate the degree to which the accuracy of tRNA selection has been optimized for maximal fitness.

No description available.