Towards understanding the mechanism of dimerisation of Saccharomyces cerevisiae eukaryotic translation initiation factor 5A
- Authors: Gentz, Petra Monika
- Date: 2008
- Subjects: Cytology Molecular biology Biochemistry Proteins -- Analysis Proteomics Polypeptides Amino acids -- Synthesis
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3992 , http://hdl.handle.net/10962/d1004052
- Description: Eukaryotic translation initiation factor 5A (eIF5A) is the only known protein to contain hypusine, formed by post-translational modification of a highly conserved lysine residue. Hypusination is essential for eIF5A function, being required for binding of a specific subset of mRNAs necessary for progression of eukaryotic cells through the G1-S checkpoint. Little structural information is available for eIF5A other than that derived from archaeal homologues. The aim of this study was to conduct structure-function studies on Saccharomyces cerevisiae (yeast) eIF5A, encoded by TIF51A. Homology models of eIF5A were generated from the Methanococcus jannaschii archaeal homologue (aIF5A) and two Leishmania eIF5As. The models, along with secondary structure predictions identified an a-helix on the C-terminal domain, unique to eukaryote eIF5A. The Neurospora crassa structural analogue, HEX-1, which dimerises in three configurations, was used to generate similar dimeric model configurations of eIF5A. A biochemical and functional analysis was used to validate the homology models of eIF5A.Since the crystal structures of aIF5A and eIF5A were solved from unhypusinated protein produced in Escherichia coli, 6 x His-tagged eIF5A (His-eIF5A) was used for biochemical analysis. This analysis revealed that eIF5A existed as a dimer in solution, dependent on the presence of the highly conserved Cys 39 residue. A yeast TIF51A/TIF51B null yeast strain, with a chromosomal copy of TIF51A under control of PGAL1, was used to confirm that HiseIF5A and selected eIF5A mutants were functional in vivo. Biochemical analysis showed that hypusinated His-eIF5A also exists as a dimer, but neither the dimerisation, nor the function of eIF5A are dependent on the presence of Cys 39. Rather they depend on the presence of hypusine (Hpu) 51 and the presence of RNA leading to the conclusion that RNA and hypusine are required for dimerisation and hence function, of native eIF5A in vivo. In contrast, a Lys 51 to Arg 51 substitution or RNase treatment of His-eIF5A produced in E. coli did not destabilize the dimeric form, suggesting different folding/dimerisation mechanisms in E. coli and yeast cells. The information obtained from the initial homology models, together with the results of the biochemical analysis was used to propose a mechanism for dimerisation of yeast eIF5A involving both hypusine and RNA.
- Full Text:
- Date Issued: 2008
- Authors: Gentz, Petra Monika
- Date: 2008
- Subjects: Cytology Molecular biology Biochemistry Proteins -- Analysis Proteomics Polypeptides Amino acids -- Synthesis
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3992 , http://hdl.handle.net/10962/d1004052
- Description: Eukaryotic translation initiation factor 5A (eIF5A) is the only known protein to contain hypusine, formed by post-translational modification of a highly conserved lysine residue. Hypusination is essential for eIF5A function, being required for binding of a specific subset of mRNAs necessary for progression of eukaryotic cells through the G1-S checkpoint. Little structural information is available for eIF5A other than that derived from archaeal homologues. The aim of this study was to conduct structure-function studies on Saccharomyces cerevisiae (yeast) eIF5A, encoded by TIF51A. Homology models of eIF5A were generated from the Methanococcus jannaschii archaeal homologue (aIF5A) and two Leishmania eIF5As. The models, along with secondary structure predictions identified an a-helix on the C-terminal domain, unique to eukaryote eIF5A. The Neurospora crassa structural analogue, HEX-1, which dimerises in three configurations, was used to generate similar dimeric model configurations of eIF5A. A biochemical and functional analysis was used to validate the homology models of eIF5A.Since the crystal structures of aIF5A and eIF5A were solved from unhypusinated protein produced in Escherichia coli, 6 x His-tagged eIF5A (His-eIF5A) was used for biochemical analysis. This analysis revealed that eIF5A existed as a dimer in solution, dependent on the presence of the highly conserved Cys 39 residue. A yeast TIF51A/TIF51B null yeast strain, with a chromosomal copy of TIF51A under control of PGAL1, was used to confirm that HiseIF5A and selected eIF5A mutants were functional in vivo. Biochemical analysis showed that hypusinated His-eIF5A also exists as a dimer, but neither the dimerisation, nor the function of eIF5A are dependent on the presence of Cys 39. Rather they depend on the presence of hypusine (Hpu) 51 and the presence of RNA leading to the conclusion that RNA and hypusine are required for dimerisation and hence function, of native eIF5A in vivo. In contrast, a Lys 51 to Arg 51 substitution or RNase treatment of His-eIF5A produced in E. coli did not destabilize the dimeric form, suggesting different folding/dimerisation mechanisms in E. coli and yeast cells. The information obtained from the initial homology models, together with the results of the biochemical analysis was used to propose a mechanism for dimerisation of yeast eIF5A involving both hypusine and RNA.
- Full Text:
- Date Issued: 2008
Utility of the cognitive assessment system (CAS) to predict reading proficiency in grade 1
- Authors: Hüttenrauch, Maria Eleonore
- Date: 2008
- Subjects: Das-Naglieri Cognitive Assessment System , Intelligence tests for preliterates , Cognition in children , Reading disability
- Language: English
- Type: Thesis , Doctoral , DPhil
- Identifier: vital:9839 , http://hdl.handle.net/10948/965 , Das-Naglieri Cognitive Assessment System , Intelligence tests for preliterates , Cognition in children , Reading disability
- Description: Reading disability, as the most commonly diagnosed learning disability, continues to pose a tenacious problem to teachers, practitioners and researchers. In an effort to understand the causes of reading disability, voluminous research has been undertaken over the past decades to pinpoint its causes or developmental stumbling blocks. One approach, the Planning, Attention, Simultaneous, and Successive processing (PASS) model, combines neuropsychological theory with elements from cognitive psychology. Based on this model, the Cognitive Assessment System (CAS), as well as methods of intervention for reading disability were developed. Although many studies have been conducted that investigated reading disability in terms of PASS cognitive processes, the final version of the CAS and its predictive utility with respect to reading disability has not been explored to date. The present study aimed to investigate the utility of the CAS, administered at the beginning of grade 1, to predict reading proficiency at the end of grade 1. The sample was comprised of 119 “average” (i.e., belonging to the general population) grade 1 students from schools of the Calgary Board of Education (CBE). The Basic Battery of the CAS was administered to the children in the sample at the beginning of grade 1, as well as four reading subtests of the Woodcock-Johnson Tests of Achievement – Third Edition (WJ-III ACH) at the end of grade 1. The WJ-III ACH yielded a cluster score for basic reading and one for reading comprehension. Correlational and regression analyses were used to address the first aim of this study, namely to explore the relationship between students’ scores on the CAS and their later reading proficiency. To this end, the children’s CAS Full Scale scores and WJ-III ACH cluster scores were subjected to a hierarchical regression analysis, whereby age, gender, and - xviii - SES were kept constant by entering them first in the equation. Next, the relationship between students’ PASS scale scores and the CAS subtest scores respectively and scores on the WJ-III ACH Basic Reading and Reading Comprehension Cluster scores was explored by means of stepwise regression analysis. To improve on the generalizability of results, the regression analyses were conducted on a randomly drawn analysis sample consisting of 80% of the sample, and cross-validated on the remaining 20% of the sample. The second aim of the present study was to ascertain whether clusters could be identified on the basis of CAS performance as well as levels of reading proficiency. To this end, the children’s CAS FS scores, PASS scale scores, and CAS subtest scores were subjected to cluster analyses. The investigation of aim 1 yielded some encouraging results, in that it was found that, together with the covariates: • The CAS FS emerged as a moderately strong predictor of both basic reading and reading comprehension; • Successive processing, in particular the Word Series subtest, significantly predicted basic reading skills; • Successive and simultaneous processing, particularly the Nonverbal Matrices and Sentence Repetition subtests, were significant predictors of reading comprehension; The second aim, which explored the relationship between patterns of CAS cognitive processes and their relationship with reading proficiency, yielded: • Two clusters with distinctly different PASS scale scores and with significant differences between their levels of reading proficiency. Higher PASS scales scores, particularly on the Attention and Planning scales, were associated with higher reading proficiency scores. • Four clusters with distinctly different CAS subtest scores that were also associated with distinctly different levels of reading performance. Good - xix - reading proficiency was associated with good CAS performance, whereas weaker reading proficiency was linked to weaker CAS performance. Biographical variables, such as age and SES were found to be related to performance on the CAS and reading proficiency, while gender did not emerge as an important predictor variable. The present study demonstrated the usefulness of the CAS, particularly its Successive and Simultaneous scales, as potential early predictor of reading disability. An exploration of the relationship between patterns of CAS cognitive processes and later reading proficiency also yielded encouraging and interesting results.
- Full Text:
- Date Issued: 2008
- Authors: Hüttenrauch, Maria Eleonore
- Date: 2008
- Subjects: Das-Naglieri Cognitive Assessment System , Intelligence tests for preliterates , Cognition in children , Reading disability
- Language: English
- Type: Thesis , Doctoral , DPhil
- Identifier: vital:9839 , http://hdl.handle.net/10948/965 , Das-Naglieri Cognitive Assessment System , Intelligence tests for preliterates , Cognition in children , Reading disability
- Description: Reading disability, as the most commonly diagnosed learning disability, continues to pose a tenacious problem to teachers, practitioners and researchers. In an effort to understand the causes of reading disability, voluminous research has been undertaken over the past decades to pinpoint its causes or developmental stumbling blocks. One approach, the Planning, Attention, Simultaneous, and Successive processing (PASS) model, combines neuropsychological theory with elements from cognitive psychology. Based on this model, the Cognitive Assessment System (CAS), as well as methods of intervention for reading disability were developed. Although many studies have been conducted that investigated reading disability in terms of PASS cognitive processes, the final version of the CAS and its predictive utility with respect to reading disability has not been explored to date. The present study aimed to investigate the utility of the CAS, administered at the beginning of grade 1, to predict reading proficiency at the end of grade 1. The sample was comprised of 119 “average” (i.e., belonging to the general population) grade 1 students from schools of the Calgary Board of Education (CBE). The Basic Battery of the CAS was administered to the children in the sample at the beginning of grade 1, as well as four reading subtests of the Woodcock-Johnson Tests of Achievement – Third Edition (WJ-III ACH) at the end of grade 1. The WJ-III ACH yielded a cluster score for basic reading and one for reading comprehension. Correlational and regression analyses were used to address the first aim of this study, namely to explore the relationship between students’ scores on the CAS and their later reading proficiency. To this end, the children’s CAS Full Scale scores and WJ-III ACH cluster scores were subjected to a hierarchical regression analysis, whereby age, gender, and - xviii - SES were kept constant by entering them first in the equation. Next, the relationship between students’ PASS scale scores and the CAS subtest scores respectively and scores on the WJ-III ACH Basic Reading and Reading Comprehension Cluster scores was explored by means of stepwise regression analysis. To improve on the generalizability of results, the regression analyses were conducted on a randomly drawn analysis sample consisting of 80% of the sample, and cross-validated on the remaining 20% of the sample. The second aim of the present study was to ascertain whether clusters could be identified on the basis of CAS performance as well as levels of reading proficiency. To this end, the children’s CAS FS scores, PASS scale scores, and CAS subtest scores were subjected to cluster analyses. The investigation of aim 1 yielded some encouraging results, in that it was found that, together with the covariates: • The CAS FS emerged as a moderately strong predictor of both basic reading and reading comprehension; • Successive processing, in particular the Word Series subtest, significantly predicted basic reading skills; • Successive and simultaneous processing, particularly the Nonverbal Matrices and Sentence Repetition subtests, were significant predictors of reading comprehension; The second aim, which explored the relationship between patterns of CAS cognitive processes and their relationship with reading proficiency, yielded: • Two clusters with distinctly different PASS scale scores and with significant differences between their levels of reading proficiency. Higher PASS scales scores, particularly on the Attention and Planning scales, were associated with higher reading proficiency scores. • Four clusters with distinctly different CAS subtest scores that were also associated with distinctly different levels of reading performance. Good - xix - reading proficiency was associated with good CAS performance, whereas weaker reading proficiency was linked to weaker CAS performance. Biographical variables, such as age and SES were found to be related to performance on the CAS and reading proficiency, while gender did not emerge as an important predictor variable. The present study demonstrated the usefulness of the CAS, particularly its Successive and Simultaneous scales, as potential early predictor of reading disability. An exploration of the relationship between patterns of CAS cognitive processes and later reading proficiency also yielded encouraging and interesting results.
- Full Text:
- Date Issued: 2008