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Exacerbation of efp- sickness in Escherichia coli by an uncharacterized RNA helicase
Title
Exacerbation of efp- sickness in Escherichia coli by an uncharacterized RNA helicase.
Creator
Wingo, Robert, Moore, Sean, Roy, Herve, Teter, Kenneth, University of Central Florida
Abstract / Description
In Escherichia coli, growth is rate-limited by translation capacity. Stalled ribosomes have profound effects on a cell such as altered mRNA abundance, decreased ribosome availability, and an imbalanced proteome. The absence of elongation factor P (EF-P), a universally conserved transpeptidation enhancer, presents an extreme example of this scenario, wherein ribosomes accumulate disproportionately onto messages that are more slowly translated and cell growth becomes notably impaired. We...
Show moreIn Escherichia coli, growth is rate-limited by translation capacity. Stalled ribosomes have profound effects on a cell such as altered mRNA abundance, decreased ribosome availability, and an imbalanced proteome. The absence of elongation factor P (EF-P), a universally conserved transpeptidation enhancer, presents an extreme example of this scenario, wherein ribosomes accumulate disproportionately onto messages that are more slowly translated and cell growth becomes notably impaired. We discovered that faster-growing cells arise spontaneously in ?efp cultures, suggesting that translation defects could be circumvented by mutating other genes. This thesis presents a genetic and biochemical analysis of a mechanism ?efp cells employ to overcome translation stress. Using a dual luciferase reporter system, we found that transpeptidation remained hindered in the faster growing ?efp cells. Whole genome sequencing of several fast-growing strains revealed mutations in a poorly characterized RNA helicase called HrpA. We determined that deletion of hrpA, or mutations at several conserved residues critical for HrpA's function, was sufficient to improve the fitness of ?efp cells. HrpA is a DEAH-box RNA helicase and represents a large class of enigmatic proteins that use ATP to restructure cellular RNAs; however, it's direct function in cellular physiology has yet to be clearly demonstrated. Several HrpA mutants were engineered to interrogate the molecular mechanism of HrpA and how its function impairs ?efp cells. Complementation in ?efp ?hrpA cells showed that a number of these mutants were unable to restore sickness, suggesting they were defective in key aspects of RNA processing. It was discovered that wild-type HrpA is associated with actively translating ribosomes and several of the inactive HrpA mutants impose substantial deleterious effects on translation and ribosome production. In sum, the work presented here describes a mechanism by which cells overcome translation stress involving a novel genetic and biochemical relationship between EF-P and HrpA.
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Date Issued
2018
Identifier
CFE0007267, ucf:52178
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0007267
It takes two to tango: the toxin-chaperone relationship
Title
It takes two to tango: the toxin-chaperone relationship.
Creator
Kellner, Alisha, Teter, Kenneth, Moore, Sean, Cole, Alexander, Harper, James, University of Central Florida
Abstract / Description
Cholera toxin (CT) enters the cell via receptor-mediated endocytosis and travels in a retrograde fashion to the endoplasmic reticulum (ER). The catalytic A1 subunit (CTA1) is then displaced from the rest of the holotoxin, unfolds, and is exported to the cytosol where it regains an active conformation for the ADP-ribosylation of its G-protein target. We have shown that the cytosolic chaperones Hsp90 and Hsc70 are required for CTA1 translocation to the cytosol. We have also shown that both are...
Show moreCholera toxin (CT) enters the cell via receptor-mediated endocytosis and travels in a retrograde fashion to the endoplasmic reticulum (ER). The catalytic A1 subunit (CTA1) is then displaced from the rest of the holotoxin, unfolds, and is exported to the cytosol where it regains an active conformation for the ADP-ribosylation of its G-protein target. We have shown that the cytosolic chaperones Hsp90 and Hsc70 are required for CTA1 translocation to the cytosol. We have also shown that both are able to independently bind and refold CTA1. Using libraries of CTA1-derived peptides, we have identified a single Hsc70 binding site, YYIYVI (CTA1 83-88), within the 192 amino acid protein, as well as two distinct Hsp90 binding sites: an N-terminal RPPDEI (CTA111-16) motif and a C-terminal LDIAPA (CTA1 153-158) motif. The LDIAPA motif is unique to CTA1, but an RPPDEI-like motif is present in four other ER-translocating ADP-ribosylating toxins: pertussis toxin, Pseudomonas aeruginosa exotoxin A, Escherichia coli heat-labile toxin, and Salmonella typhimurium ADP-ribosylating toxin. Using site-directed mutagenesis to further investigate the RPPDEI motif, we found that a modification of either proline residue blocks CTA1 translocation to the cytosol. Our work has identified, for the first time, specific amino acid sequences that are recognized by Hsp90/Hsc70 and are essential for toxin translocation from the ER to the cytosol. CT does not require prolyl isomerases for cellular activity, as is the case for Hsp90-dependent endosome-translocating toxins. We therefore hypothesize that the one or both of the prolines within the RPPDEI motif of CTA1 undergo an isomerization event as CTA1 unfolds in the ER. Furthermore, we predict that the trans- to cis- conformational change of proline(s) is the molecular determinate for the atypical Hsp90 interaction observed with CTA1 and related toxins. Additionally, we have identified Hsp90 and other host factors required for the translocation of pertussis toxin.
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Date Issued
2019
Identifier
CFE0007661, ucf:52500
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0007661
Behavioral and disease ecology of Gopher Tortoises (Gopherus polyphemus) post exclusion and relocation with a novel approach to homing determination
Title
Behavioral and disease ecology of Gopher Tortoises (Gopherus polyphemus) post exclusion and relocation with a novel approach to homing determination.
Creator
Napier, Johnathan, Savage, Anna, Moore, Sean, Vonkalm, Laurence, Fedorka, Kenneth, University of Central Florida
Abstract / Description
In the wake of human expansion, relocations and the loss of habitat can be stressful to an organism, plausibly leading to population declines. The gopher tortoise (Gopherus polyphemus) is a keystone species that constructs burrows it shares with 362 commensal species. Frequent exclusions and relocations and long generation times have contributed to G. polyphemus being State-designated as Threatened in Florida. Prior studies have indicated that G. polyphemus may possess homing behavior and...
Show moreIn the wake of human expansion, relocations and the loss of habitat can be stressful to an organism, plausibly leading to population declines. The gopher tortoise (Gopherus polyphemus) is a keystone species that constructs burrows it shares with 362 commensal species. Frequent exclusions and relocations and long generation times have contributed to G. polyphemus being State-designated as Threatened in Florida. Prior studies have indicated that G. polyphemus may possess homing behavior and thus be able to counteract stressors due to relocation and exclusion. I radiotracked a cohort of G. polyphemus for 11 months following excavation, relocation, and exclusion due to a pipeline construction project. In conjunction with analyzing G. polyphemus movement patterns post-release, I developed novel statistical methodologies with broad application for movement analysis and compared them to traditional analyses. I evaluated habitat usage, burrowing behavior, movements, growth, and disease signs among control versus relocated and excluded individuals and among sexes and size classes, forming predictors for behavior and disease risk. I found statistical support that my new methodology is superior to previous statistical tests for movement analyses. I also found that G. polyphemus engages in homing behavior, but only in males. Behavioral differences were also found between the sexes with respect to burrowing behavior. Overall health, disease prevalence, and immune response were unaffected by relocation and exclusion, nor were they statistically correlated. Signs were unreliable as etiological agents, outperformed by serological detection. I determined that the Sabal Trail pipeline as a potential stressor did not affect movement behavior, homing, nor the disease/immune profile of G. polyphemus in this study.
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Date Issued
2018
Identifier
CFE0007581, ucf:52581
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0007581
The role of a highly conserved eubacterial ribosomal protein in translation quality control
Title
The role of a highly conserved eubacterial ribosomal protein in translation quality control.
Creator
Naganathan, Anusha, Moore, Sean, Cole, Alexander, Teter, Kenneth, Roy, Herve, Koculi, Eda, University of Central Florida
Abstract / Description
The process of decoding is the most crucial determinant of the quality of protein synthesis. Ribosomal protein L9 was first implicated in decoding fidelity when a mutant version of L9 was found to increase the translation of a T4 phage gene. Later studies confirmed that the absence of L9 leads to increased translational bypassing, frameshifting, and stop codon readthrough. L9 is part of the large subunit of the prokaryotic ribosome and is located more than 90 (&)#197; from the site of...
Show moreThe process of decoding is the most crucial determinant of the quality of protein synthesis. Ribosomal protein L9 was first implicated in decoding fidelity when a mutant version of L9 was found to increase the translation of a T4 phage gene. Later studies confirmed that the absence of L9 leads to increased translational bypassing, frameshifting, and stop codon readthrough. L9 is part of the large subunit of the prokaryotic ribosome and is located more than 90 (&)#197; from the site of decoding, making it difficult to envision how it might affect decoding and reading frame maintenance. Twenty years after the identification of L9's putative function, there is no mechanism for how a remotely located L9 improves translation fidelity. This mystery makes our picture of translation incomplete. Despite the high conservation of L9 in eubacteria, E.coli lacking L9 does not exhibit any obvious growth defects. Thus, the evolutionary advantage conferred by L9 in bacteria is masked under laboratory conditions. In order to uncover unique L9-dependent conditions, a library of E. coli mutants was screened to isolate those that rely on L9 for fitness. Interestingly, factors found to be synergistic with L9 had no known role in fidelity. Six independent mutants were isolated, each exhibiting a severe growth defect that is partially suppressed in the presence of L9. One class of L9-dependent mutations was present in an essential ribosome biogenesis factor, Der. Der's established function is in the maturation of the large ribosomal subunit. The identified mutations severely impaired the GTPase activity of Der. Interestingly, L9 did not directly compensate for the defective GTPase activity of mutant Der. The second class of L9-dependent mutations was present in EpmA and EpmB, factors required to post-translationally modify elongation factor, EF-P. EF-P's established function is in the translation of poly-proline containing proteins. EF-P deficient cells were nearly inviable in the absence of L9; however, L9 did not directly influence poly-proline translation. Therefore, in each case, L9 improved cell health without altering the activity of either Der or EF-P. Remarkably, the der mutants required only the N domain of L9, whereas the absence of active EF-P required full-length, wild-type L9 for growth complementation. Thus, each mutant class needed a different aspect of L9's unique architecture. In cells lacking either active EF-P or Der, there was a severe deficiency of 70S ribosomes and the indication of small subunit maturation defects, both of which worsened upon L9 depletion. These results strongly suggest that L9 plays a role in improving ribosome quality and abundance under certain conditions.Overall, the genetic screen lead to the discovery that bacteria need L9 when either of two important translation factors (Der or EF-P) is inactivated. This work has characterized the physiological requirement for L9 in each case and offers a new insight into L9's assigned role in translation fidelity.
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Date Issued
2015
Identifier
CFE0005674, ucf:50169
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0005674
Middle Grades Science in Florida: A Comparison of Student Achievement in Comprehensive and Subject-specific Science Courses 2013-2017
Title
Middle Grades Science in Florida: A Comparison of Student Achievement in Comprehensive and Subject-specific Science Courses 2013-2017.
Creator
Moore, Kenneth, Taylor, Rosemarye, Baldwin, Lee, Storey, Valerie A., Gao, Su, University of Central Florida
Abstract / Description
As with U.S. student achievement on national and international science assessments, Florida's 8th grade student achievement on the 2013(-)2017 8th grade Florida Comprehensive Assessment Test (FCAT) 2.0 Science/Statewide Science Assessment (SSA) was stagnant. To break this stagnation, many Florida school districts have changed middle grades science course offerings from traditional, subject-specific, discipline-based, layered, or field-specific science courses to comprehensive, integrated,...
Show moreAs with U.S. student achievement on national and international science assessments, Florida's 8th grade student achievement on the 2013(-)2017 8th grade Florida Comprehensive Assessment Test (FCAT) 2.0 Science/Statewide Science Assessment (SSA) was stagnant. To break this stagnation, many Florida school districts have changed middle grades science course offerings from traditional, subject-specific, discipline-based, layered, or field-specific science courses to comprehensive, integrated, spiraled, interdisciplinary, multidisciplinary, thematic, or general science courses. There was a lack of research showing if either type of science course improved student achievement on standardized science assessments. Controlling for school district student population, low socio-economic status (SES) student percentage, and English learner (EL) percentage, this study compared the 2013(-)2017 8th grade FCAT 2.0 Science/SSA school district mean scale scores of two groups of school districts: those that offered comprehensive science courses and those that offered subject-specific science courses. Scores for three student groups were analyzed: all students, low SES students, and ELs. No statistically significant differences were found in school district mean scale scores or pass rates between the two school district groups. The comprehensive group mean scale scores were numerically higher, while the subject-specific group mean pass rates were numerically higher. The subject-specific group had statistically significantly higher raw scores for life science and physical science. The comprehensive group had wider dispersions of mean scale scores and pass rates, suggesting inconsistencies in implementation of comprehensive science courses. The primary implication of this study is that educational leaders should not expect to improve student science achievement simply by changing the type of science course offering. Changes should be made with consideration to student needs, school district demographics, teacher professional development and support, course structure and coherence with standards, and the need for flexibility in teacher assignments.
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Date Issued
2018
Identifier
CFE0007410, ucf:52710
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0007410