No description available.

Scaffolding proteins control the localization of protein kinases. During translation, the scaffolding proteins eIF4G and DAP5 recruit the Ser/Thr kinase Mnk1 to phosphorylate the mRNA cap-binding protein eIF4E and modulate translation. Biochemical deletion analysis previously showed that the interaction between Mnk1 and eIF4G/DAP5 is mediated by N-terminal residues in Mnk1 and C-terminal residues in eIF4G/DAP5. Using X-ray crystallography I have determined the structure (1.5 A) of the C-terminal domain of DAP5 (DAP5C). This structure reveals that DAP5C contains two atypical HEAT domains similar to the ones seen previously in the structure of the C-terminal region of eIF4G (4GC). Using ITC I showed that the Kd for the interaction between the N-terminus ofMnk1 and 4GCIDAPSC is 20 muM and 10 muM, respectively. Using NMR chemical shifts we have mapped the residues on both Mnk1 and 4GC/DAP5C which are important for maintaining this interaction. Finally, using SAXS a low resolution configuration of the hMnk1-4GC complex was modeled. It is hoped that an understanding of the structural basis for the recruitment of protein kinases to their sites of action will allow the design of small-molecule compounds that can be used to modulate the location of the kinase and hence its activity.

Thesis (M.Sc.). / Written for the Dept. of Biochemistry. Title from title page of PDF (viewed 2008/05/29). Includes bibliographical references.

No description available.

The process of translation initiation is the most highly regulated step of protein synthesis. In bacteria, three initiation factors (IF1, IF2 and IF3) play crucial roles during initiation. IF3 acts as an anti-association factor for the two ribosomal subunits. Eubacterial IF3 also permits initiator tRNA (i-tRNA) selection at the P site of the ribosome. Two features of i-tRNA, i. e. the characteristic 3GC base pairs in the anticodon stem and the cognate interaction of the anticodon sequence with the initiation codon of the mRNA contribute to IF3 based selection and/or proofreading. However, the exact mechanism of this discrimination and the contribution of the individual domains towards this process of selection/ proofreading are unclear. Further, there are exceptional instances in the natural world where either the codon-anticodon interaction or the anticodon stem composition deviates from the norm. For instance, in mammalian mitochondria, non-AUG codons such as AUU and AUA are present in the genome although they are notoriously poor initiation codons. In addition, some species of Mycoplasma have i-tRNAs with variations in the typically conserved 3GC base pairs of the anticodon stem. In this study, we have investigated the mechanism of proofreading activity of IF3 of E. coli, mitochondrial and mycoplasmal origins. Part I: Proofreading function of IF3 in E. coli IF3 is composed of N and C terminal domains joined by a flexible linker region. By means of complete and partial IF3 knockouts, we show that the C-terminal domain (CTD) is essential for the survival of E. coli while the N-terminal (NTD) is required for cellular fitness. Using reporter assays, we have established the role of the NTD in proofreading, while polysome profile analyses reaffirm that the CTD alone can bind to the 30S and carry out ribosome anti-association. Therefore, we show that the CTD is the ribosome binding and anti-association domain, while the NTD is the major proofreading domain. Unpublished cryoEM structures from Prof. Ramakrishnan’s lab indicate that the NTD of IF3 pushes the i-tRNA at its elbow and helps in P site accommodation of the i-tRNA. We propose that when the codon-anticodon interaction is non-cognate or if the 3GC base pairs of the anticodon stem are not intact, then the dynamic action of the NTD destabilises the tRNA at the P site and leads to its rejection. Part II: Proofreading function of mitochondrial IF3 (IF3mt) Of the 13 protein-coding genes in mammalian mitochondria, 3 utilise the non-canonical AUA codon and one utilises the non-canonical start codon AUU. Since IF3mt does not possess many of the generally conserved residues implicated in proofreading, we decided to characterise the proofreading function of IF3mt and its role in initiation with non-canonical start codons. Structurally, IF3mt is similar to EcoIF3 with its N and C terminal domains joined by a linker region. However, IF3mt additionally possesses N- and C-terminal extensions which are generally disordered in structure. In vivo studies of mitochondrial translation factors have been mired by the lack of methodologies to manipulate mitochondria. We have developed an E. coli strain to study the proofreading functions of mitochondrial IF3 (IF3mt) with the help of reporter genes. Consistent with its function in mitochondria, IF3mt allowed promiscuous initiation from non-AUG codons. However, IF3mt avoided initiation with i-tRNAs lacking evolutionarily conserved 3GC pairs in anticodon stems. Interestingly, expression of IF3mt N-terminal domain or IF3mt devoid of its typical N-, and C-terminal extensions significantly improved its proofreading activity. Our immunoblot assays from polysome profile fractions indicate that the IF3mt derivative lacking extensions is capable of superior 30S ribosome binding. The two derivatives of IF3mt missing the Next (IF3mtΔNext) or both the Next and Cext (IF3mtΔNextCext) display an affinity for the 50S ribosome. We propose that the extensions of IF3mt may have evolved to reduce the affinity of IF3mt to the ribosome and thereby permit initiation with non-canonical start codons like AUU and AUA. Our studies suggest that E. coli provides an excellent heterologous model to study distinctive features of mitochondrial factors. Part III: Fidelity of translation initiation in mycoplasma One of the many singular features of mycoplasma is the presence of many anticodon stem variants of the i-tRNA across different species. In general, i-tRNAs are characterized by the presence of the typical feature of the conserved 3 consecutive GC base pairs (GC/GC/GC) in the anticodon stem. However, many mycoplasmal species have i-tRNAs with AU/GC/GC, GC/GC/GU or AU/GC/GU sequences. Interestingly, the mycoplasmal species which harbour the AU/GC/GU i-tRNA are also human pathogens. Therefore, we decided to investigate whether these organisms possess any unique features to accommodate the i-tRNA variants, by investigating the usage of Shine Dalgarno sequences and by carrying out multiple sequence alignments of genes encoding initiation factors, ribosomal proteins S9 and S13 and 16S rRNA. Since IF3 plays a crucial role in i-tRNA selection, we carried out computational analysis of mycoplasmal IF3 sequences, which revealed many interesting features. Most striking amongst them was the variation of the highly conserved R at position 131 in some species. Interestingly, these were the very mycoplasmal species which possessed the anticodon stem variant AU/GC/GU, suggesting a strong correlation between these two features. It is known that the R131P mutation of EcoIF3 is characterised by an enormous loss of proofreading activity. It seemed unusual that such compromised proofreading would be tolerated in the cell, so we decided to investigate other components of the translational machinery as well. The C-terminal SKR tail of the ribosomal protein S9, which contacts the P-site tRNA, is highly conserved across bacteria. Analysis of the C-terminal sequences of S9 proteins in various mycoplasmal species revealed a surprising variation- the presence of a TKR tail in strains with the AU/GC/GU tRNA. In this study we have investigated the co-occurrence of S9 and IF3 variations in i-tRNA selection in E. coli. We see that the R131P polymorphism of IF3 leads to a tremendous loss of proofreading, but this loss is significantly tempered by the presence of the S9 TKR variation. Our bioinformatics studies revealed that the mycoplasmal species which are sustained on AU/GC/GU i-tRNAs also tend to use a higher percentage of non-AUG codons. By means of our reporter assays in E. coli, we have shown once again that the R131P polymorphism of IF3 leads to a tremendous increase in initiation with the non-canonical start codon AUA, but this increase is significantly tempered by the presence of the S9 TKR variation.

<p>Initiation is the first step in protein biosynthesis representing a fundamental event in cell life which determines fidelity, efficiency and regulation of gene expression. In addition to the ribosome and mRNA, three protein factors IF1, IF2 and IF3 are involved in the initiation of translation in prokaryotes. Several minor functions have been attributed to the smallest of these factors, IF1. However, the main function of IF1 remains to be elucidated.</p><p>In order to investigate the role of this protein in the initiation process we have mutated the corresponding gene infA. Using a high-copy plasmid and site-directed mutagenesis, the six arginine residues of IF1 were separately altered to leucine or aspartate. Another set of plasmid-encoded IF1 mutants with a cold-sensitive phenotype was collected using localized random mutagenesis. This strategy was followed by deletion of the chromosomal infA gene. All variants with a mutated infA gene on a plasmid and a deletion of the chromosomal infA copy were viable, except for an R65D alteration. Several of the mutated infA genes were successfully recombined into the chromosome thereby replacing the wild-type allele. Some of these mutants displayed reduced growth rates and a partial cold-sensitive phenotype.</p><p>The influence of the leucine group of mutants in IF1 on the expression of two reporter genes with different initiation and/or +2 codons has been investigated. Our results do not indicate any involvement of IF1 in recognition of the +2 codon immediately following the start codon, thus representing the A-site. In addition, this group of mutants has no changed efficiency of decoding at the near-cognate initiation codons UUG and GUG. However, one cold-sensitive IF1 mutant shows a general overexpression of both reporter genes, in particular at low temperatures. Overall, the results do not support the hypothesis that IF1 could possess codon discriminatory functions while blocking the A-site of the ribosome.</p><p>In this study we also identify that IF1 has RNA chaperone activity both in vitro and in vivo. The chaperone assays are based on splicing of the group I intron in the thymidylate synthase gene (td) from phage T4. Some of the IF1 mutant variants are more active as RNA chaperones than the wild-type. Both wild-type IF1 and mutant variants bind with high affinity to RNA in a band-shift assay. It is suggested that the RNA chaperone activity of IF1 contributes to RNA rearrangements during the early phase of translation initiation.</p>

Initiation is the first step in protein biosynthesis representing a fundamental event in cell life which determines fidelity, efficiency and regulation of gene expression. In addition to the ribosome and mRNA, three protein factors IF1, IF2 and IF3 are involved in the initiation of translation in prokaryotes. Several minor functions have been attributed to the smallest of these factors, IF1. However, the main function of IF1 remains to be elucidated. In order to investigate the role of this protein in the initiation process we have mutated the corresponding gene infA. Using a high-copy plasmid and site-directed mutagenesis, the six arginine residues of IF1 were separately altered to leucine or aspartate. Another set of plasmid-encoded IF1 mutants with a cold-sensitive phenotype was collected using localized random mutagenesis. This strategy was followed by deletion of the chromosomal infA gene. All variants with a mutated infA gene on a plasmid and a deletion of the chromosomal infA copy were viable, except for an R65D alteration. Several of the mutated infA genes were successfully recombined into the chromosome thereby replacing the wild-type allele. Some of these mutants displayed reduced growth rates and a partial cold-sensitive phenotype. The influence of the leucine group of mutants in IF1 on the expression of two reporter genes with different initiation and/or +2 codons has been investigated. Our results do not indicate any involvement of IF1 in recognition of the +2 codon immediately following the start codon, thus representing the A-site. In addition, this group of mutants has no changed efficiency of decoding at the near-cognate initiation codons UUG and GUG. However, one cold-sensitive IF1 mutant shows a general overexpression of both reporter genes, in particular at low temperatures. Overall, the results do not support the hypothesis that IF1 could possess codon discriminatory functions while blocking the A-site of the ribosome. In this study we also identify that IF1 has RNA chaperone activity both in vitro and in vivo. The chaperone assays are based on splicing of the group I intron in the thymidylate synthase gene (td) from phage T4. Some of the IF1 mutant variants are more active as RNA chaperones than the wild-type. Both wild-type IF1 and mutant variants bind with high affinity to RNA in a band-shift assay. It is suggested that the RNA chaperone activity of IF1 contributes to RNA rearrangements during the early phase of translation initiation.

Eukaryotic initiation factor-4E (eIF4E) controls the rate of cap-dependent translation initiation and is in turn exquisitely regulated by 4E-BPs. 4E-BP2 binds eIF4E with the highest affinity and is implicated in cancer, and metabolic and neurological disorders. Herein we use NMR, ITC and fluorescence to characterize 4E-BP2 structural and binding properties. Isolated 4E-BP2 is intrinsically disordered, but possesses some transient secondary structural propensities. eIF4E, however, is folded but has a disordered N-terminus. The eIF4E:4E-BP2 interaction is tight (Kd = 10-9 nM) and involves 4E-BP2 C-terminal and canonical binding regions, and the disordered eIF4E N-terminus. 4E-BP2 remains largely disordered upon binding to eIF4E. Noteworthy, high affinity interactions are not necessarily mediated by static structures, and 4E-BP2 binding is not the simple “disorder-to-order” transition observed in many interactions involving disordered proteins. This study offers molecular insights into 4E-BP2 functionality, and lays a foundation for development of novel therapies for cancer and neurological disorders.

Eukaryotic initiation factor-4E (eIF4E) controls the rate of cap-dependent translation initiation and is in turn exquisitely regulated by 4E-BPs. 4E-BP2 binds eIF4E with the highest affinity and is implicated in cancer, and metabolic and neurological disorders. Herein we use NMR, ITC and fluorescence to characterize 4E-BP2 structural and binding properties. Isolated 4E-BP2 is intrinsically disordered, but possesses some transient secondary structural propensities. eIF4E, however, is folded but has a disordered N-terminus. The eIF4E:4E-BP2 interaction is tight (Kd = 10-9 nM) and involves 4E-BP2 C-terminal and canonical binding regions, and the disordered eIF4E N-terminus. 4E-BP2 remains largely disordered upon binding to eIF4E. Noteworthy, high affinity interactions are not necessarily mediated by static structures, and 4E-BP2 binding is not the simple “disorder-to-order” transition observed in many interactions involving disordered proteins. This study offers molecular insights into 4E-BP2 functionality, and lays a foundation for development of novel therapies for cancer and neurological disorders.

Thesis (Ph. D.)--Case Western Reserve University, 2005. / [School of Medicine Molecular Virology Program. Includes bibliographical references. Available online via OhioLINK's ETD Center.