Browsing by Author "E. Stuart Maxwell, Committee Member"

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Cis-acting Elements Important for Potato Virus X Minus-strand RNA Synthesis In Vitro and In Vivo
(2007-02-27) Hu, Bin; Paul Wollenzien, Committee Member; Cynthia L. Hemenway, Committee Chair; Barbara Sherry, Committee Member; E. Stuart Maxwell, Committee Member
In order to identify cis-acting elements required for Potato virus X (PVX) minus-strand RNA synthesis, replication in in vitro and in vivo systems was compared. Specifically, RNA dependent RNA polymerase activity using a 850 nt template in PVX infected tobacco plant extract and using infectious full-length transcripts in tobacco protoplasts was analyzed. To facilitate quantitation of results from the in vitro system, the RdRp assay was optimized in several ways. Initial experiments showed that processing of extracts from fresh plant tissue was optimal for the in vitro RdRp assay. Purification of nuclease Bal31 with stable RNase activity was critical for consistency in making and assaying template-dependent plant extracts. Optimal salt concentrations, reaction volume, incubation time, and template concentration conditions were found to ensure template specificity and quantifiable product levels for PVX RdRp. Comparison of data obtained with this optimal extract and the protoplast system showed that the conserved hexanucleotide element and conformation of stem-loop 3 are required for minus-strand RNA synthesis both in vitro and in vivo. More strikingly, we found that long-distance RNA-RNA interactions between conserved internal elements and the hexanucleotide element are required for optimal minus-strand RNA synthesis both in vitro and in vivo. In addition, multiple internal elements can serve as interaction partners. Thus, similar to plus-strand RNA synthesis, PVX minus-strand RNA synthesis requires local elements and long-distance RNA-RNA interactions. A model for RNA synthesis is proposed in which both termini of the genome are paired with internal elements.
Interactions in the Active Site Loops are Important for Maintaining the Active Site Environment of (Pro)caspase-3
(2004-04-16) Feeney, Brett; E. Stuart Maxwell, Committee Member; Dr. A. Clay Clark, Committee Chair; Carla Mattos, Committee Member
Apoptosis is obligatory to development and in maintaining the vital balance between cell growth and death. Commitment to apoptosis involves a proteolytic cascade by a family of cysteine proteases named caspases. Caspases are split it into two general classes, those involved in the proinflammatory response and those involved in apoptosis. Of those involved in apoptosis, there are two further subdivisions, the apical caspases and the executioner caspases. The irreversible commitment to apoptosis involves activation of executioner caspases, namely caspase-3. Procaspase-3 exists in the cell as a dimeric zymogen, where upon limited proteolytic cleavage at specific aspartate residues, it is activated and apoptosis results. We have previously shown the (pro)caspase-3 undergoes pH dependent conformational changes monitored by fluorescence emission (Bose, et al 2003). In this study, we unambiguously assign dimer dissociation to one of the pH dependent transitions observed in this assay by using size exclusion chromatography. We have also examined the effects of breaking specific salt bridges and hydrogen bonds by mutating residues in context of caspase-3, an inactive procaspase-3(C163S) and an uncleavable procaspase-3(D3A). We show that there are a number of stabilizing contacts that are required in order to ensure proper processing during maturation, ensure enzyme fidelity and maintain overall structure. At present, the function of the prodomain of executioner caspases has elucidated researchers. We have also hypothesized that the effector caspase prodomain may have some role as an intramolecular chaperone during maturation. We show that the caspase-3 prodomain does play a role in pH dependent folding of the (pro)caspase-3 dimer. Salt has also been well described as having effects on caspase-3 activity and stability. There is a lack of knowledge as to the direct effects that different ions have on both procaspase-3 zymogen and mature caspase-3. In this study, we describe direct effects of different cations on (pro)caspase-3 activity and active site environment. We also study stabilizing effects of salt on (pro)caspase-
Is Mth1483p a subunit of RNase P in Methanothermobacter thermoautotrophicus?
(2004-12-01) Barnes, Jeffrey Paul; James W. Brown, Committee Chair; Amy Grunden, Committee Member; E. Stuart Maxwell, Committee Member
The buoyant density of M. thermoautotrophicus RNase P was recently determined to be 1.42 g/mL. This corresponds to RNA to protein ration of 0.96, indicating that ~93 kDa of protein is present in the holoenzyme. With only 70 kDa of protein identified thus far, additional protein subunits may exist. Recent PSI-Blast searches identified ORF 1483 of M. thermoautotrophicus as a homolog of Rpp25, a human RNase P subunit. Mth1483 and Rpp25 were also identified as members of the Alba family of proteins whose members participate in both DNA packaging and RNA interactions. This suggested the possibility that Mth1483p may be a subunit of RNase P in M. thermoautotrophicus. To evaluate this, polyclonal antisera was generated against Mth1483p. Western blot analysis of glycerol gradient purified M. thermoautotrophicus RNase P showed that Mth1483p did not co-purify with RNase P activity. Also, protein-A agarose beads cross-linked with Mth1483 antibody did not immunoprecipitate RNase P activity. There is no evidence, then, that Mth1483p is an RNase P subunit.
Post-transcriptional Modifications of the tRNA Anticodon Stem and Loop (ASL) Affect the Ability of tRNA to Bind Synonymous Codons.
(2009-08-17) Gustilo, Estella M.; Paul F. Agris, Committee Chair; E. Stuart Maxwell, Committee Member; Linda Spremulli, Committee Member; Paul Wollenzien, Committee Member
The Genetic Code is arranged into sixteen codon boxes, where the four codons in each box are similar in their first two letters but differ at the third position (the wobble position). In the universal Genetic Code, each amino acid, except for Tryptophan and Methionine that have one codon each, is encoded by two to six codons (two to six-fold degenerate). There are fewer tRNA species than codons; therefore, a tRNA species can recognize more than one codon. This flexibility in recognition resides in the third position of the codon:anticodon pairing, the wobble position. Codon recognition by tRNA is dependent on the anticodon loop. The sequence of the anticodon (tRNA positions 34, 35, and 36) does not necessarily predict codon binding according to Watson-Crick rules. In all organisms, post-transcriptional modifications occur quite extensively and of great variety at the anticodon loop. These modifications, usually found on the nucleosides in tRNA position 34 (the wobble position) and position 37, direct the tRNAÃ¢â‚¬â„¢s ability to read codons accurately and efficiently. Just as the types of modifications are diverse, the abilities of modifications to recognize codons also vary. When a particular amino acid is encoded by an entire codon box, such as four-fold degenerate Valine, we show that a tRNA species with a specific modification can read all four codons of that box. The modification 5-oxyacetic acid (cmo5) at the wobble position of tRNAVal (tRNAVal-cmo5U34) allows cmo5U34 to recognize U, A, G, and perhaps C. In instances where the difference between the codes of two amino acids resides only in the third letter of their codons (2-fold degenerate codons in a split box), modifications at the wobble position of the anticodon restrict codon recognition to the two codons specific for that 2-fold degenerate amino acid. For example, Lysine has two codons (AAA and AAG) that share a codon box with Asparagine codons (AAU and AAC). The modified nucleoside 5-methoxycarbonylmethyl-2-thiouridine at the wobble position of human tRNA (tRNALys-mcm5s2U34) confers this tRNAÃ¢â‚¬â„¢s ability to restrict codon recognition to the two Lysine codons only. Similar to the cytoplasmic tRNAs, mitochondrial tRNAs also contain posttranscriptional modifications. The mitochondria deviate from the universal Genetic Code in that it uses the universal Isoleucine codon AUA to decode Methionine. In all organisms, there are two Methionine tRNAs: an initiator tRNAMet and an elongator tRNAMet. Mitochondria, however, have but one tRNAMet that acts as both initiator and elongator, has characteristics of both types of tRNAMet, and decodes AUG and AUA in the aminoacyl- (entry or A)-site and the peptidyl (P)-site of the ribosome. The human mitochondrial tRNAMet is modified with a 5-formyl-group at the wobble position cytidine-34 (hmtRNAMetf5C34). This modification allows the hmtRNAMet f5C34 to expand codon recognition to include AUA in translating Methionine. At times, the mitochondria use Isoleucine codons AUU and AUC to initiate translation. Surprisingly, the 5-formyl modification of hmtRNAMet-f5C34 also allows codon-reading expansion at the P-site to include the entire codon box AUN.