A genetic and biochemical analysis of pre-mRNA splicing in Saccharomyces cerevisiae

Pre-mRNA splicing requires interaction of cis- acting intron sequences with trans -acting factors: proteins and small nuclear ribonucleoproteins (snRNPs). The assembly of these factors into a large complex, the spliceosome, is essential for the subsequent two step splicing reaction. First, the 5' splice site is cleaved and free exon 1 and a lariat intermediate (intron- exon2) form. In the second reaction the 3' splice site is cleaved the exons ligated and lariat intron released. A combination of genetic and biochemical techniques have been used here to study pre-mRNA splicing in yeast.

Yeast introns have three highly conserved elements. We made point mutations within these elements and found that most of them affect splicing efficiency in vivo and in vitro, usually by inhibiting spliceosome assembly.

To study trans -acting splicing factors we generated and screened a bank of temperature- sensitive (ts) mutants. Eleven new complementation groups (prp17 to prp27) were isolated. The four phenotypic classes obtained affect different steps in splicing and accumulate either: 1) pre-mRNA, 2) lariat intermediate, 3) excised intron or 4) both pre-mRNA and intron. The latter three classes represent novel phenotypes. The excised intron observed in one mutant: prp26 is stabilized due to protection in a snRNP containing particle. Extracts from another mutant: prpl8 are heat labile and accumulate lariat intermediate and exon 1. This is especially interesting as it allows analysis of the second splicing reaction. In vitro complementation of inactivated prp18 extracts does not require intact snRNPs. These studies have also shown the mutation to be in a previously unknown splicing protein. A specific requirement for A TP is also observed for the second step of splicing. The PRP 18 gene has been cloned and its polyadenylated transcript identified.

A Detailed Analysis of Transcriptional Regulators Affecting the Saccharomyces cerevisiae Heat-shock Gene SSA1

The yeast Saccharomyces cerevisiae contains a family of hsp70 related genes. One member of this family, SSA1, encodes a 70kD heat-shock protein which in addition to its heat inducible expression has a significant basal level of expression. The first 500 bp upstream of the SSA1 start point of transcription was examined by DNAse I protection analysis. The results reveal the presence of at least 14 factor binding sites throughout the upstream promoter region. The function of these binding sites has been examined using a series of 5' promoter deletions fused to the recorder gene lacZ in a centromere-containing yeast shuttle vector. The following sites have been identified in the promoter and their activity in yeast determined individually with a centromere-based recorder plasmid containing a truncated CYC1 /lacZ fusion: a heat-shock element or HSE which is sufficient to convey heat-shock response on the recorder plasmid; a homology to the SV40 'core' sequence which can repress the GCN4 recognition element (GCRE) and the yAP1 recognition element (ARE), and has been designated a upstream repression element or URE; a 'G'-rich region named G-box which can also convey heatshock response on the recorder plasmid; and a purine-pyrimidine alternating sequence name GT-box which is an activator of transcription. A series of fusion constructs were made to identify a putative silencer-like element upstream of SSA1. This element is position dependent and has been localized to a region containing both an ABF1 binding site and a RAP1 binding site. Five site-specific DNA-binding factors are identified and their purification is presented: the heat-shock transcription factor or HSTF, which recognizes the HSE; the G-box binding factor or GBF; the URE recognition factor or URF; the GT-box binding factor; and the GC-box binding factor or yeast Sp1.

Proteomic analysis of the Cdc48/Ubx network identifies a role for Ubx2 in the regulation of lipid biosynthesis

Cdc48/p97 is an essential, highly abundant hexameric member of the AAA (ATPase associated with various cellular activities) family. It has been linked to a variety of processes throughout the cell but it is best known for its role in the ubiquitin proteasome pathway. In this system it is believed that Cdc48 behaves as a segregase, transducing the chemical energy of ATP hydrolysis into mechanical force to separate ubiquitin-conjugated proteins from their tightly-bound partners.

Current models posit that Cdc48 is linked to its substrates through a variety of adaptor proteins, including a family of seven proteins (13 in humans) that contain a Cdc48-binding UBX domain. As such, due to the complexity of the network of adaptor proteins for which it serves as the hub, Cdc48/p97 has the potential to exert a profound influence on the ubiquitin proteasome pathway. However, the number of known substrates of Cdc48/p97 remains relatively small, and smaller still is the number of substrates that have been linked to a specific UBX domain protein. As such, the goal of this dissertation research has been to discover new substrates and better understand the functions of the Cdc48 network. With this objective in mind, we established a proteomic screen to assemble a catalog of candidate substrate/targets of the Ubx adaptor system.

Here we describe the implementation and optimization of a cutting-edge quantitative mass spectrometry method to measure relative changes in the Saccharomyces cerevisiae proteome. Utilizing this technology, and in order to better understand the breadth of function of Cdc48 and its adaptors, we then performed a global screen to identify accumulating ubiquitin conjugates in cdc48-3 and ubxΔ mutants. In this screen different ubx mutants exhibited reproducible patterns of conjugate accumulation that differed greatly from each other, pointing to various unexpected functional specializations of the individual Ubx proteins.

As validation of our mass spectrometry findings, we then examined in detail the endoplasmic-reticulum bound transcription factor Spt23, which we identified as a putative Ubx2 substrate. In these studies ubx2Δ cells were deficient in processing of Spt23 to its active p90 form, and in localizing p90 to the nucleus. Additionally, consistent with reduced processing of Spt23, ubx2Δ cells demonstrated a defect in expression of their target gene OLE1, a fatty acid desaturase. Overall, this work demonstrates the power of proteomics as a tool to identify new targets of various pathways and reveals Ubx2 as a key regulator lipid membrane biosynthesis.

Role of feedback and dynamics in a gene regulatory network

Cells exhibit a diverse repertoire of dynamic behaviors. These dynamic functions are implemented by circuits of interacting biomolecules. Although these regulatory networks function deterministically by executing specific programs in response to extracellular signals, molecular interactions are inherently governed by stochastic fluctuations. This molecular noise can manifest as cell-to-cell phenotypic heterogeneity in a well-mixed environment. Single-cell variability may seem like a design flaw but the coexistence of diverse phenotypes in an isogenic population of cells can also serve a biological function by increasing the probability of survival of individual cells upon an abrupt change in environmental conditions. Decades of extensive molecular and biochemical characterization have revealed the connectivity and mechanisms that constitute regulatory networks. We are now confronted with the challenge of integrating this information to link the structure of these circuits to systems-level properties such as cellular decision making. To investigate cellular decision-making, we used the well studied galactose gene-regulatory network in \textit{Saccharomyces cerevisiae}. We analyzed the mechanism and dynamics of the coexistence of two stable on and off states for pathway activity. We demonstrate that this bimodality in the pathway activity originates from two positive feedback loops that trigger bistability in the network. By measuring the dynamics of single-cells in a mixed sugar environment, we observe that the bimodality in gene expression is a transient phenomenon. Our experiments indicate that early pathway activation in a cohort of cells prior to galactose metabolism can accelerate galactose consumption and provide a transient increase in growth rate. Together these results provide important insights into strategies implemented by cells that may have been evolutionary advantageous in competitive environments.

Engineering enzyme systems by recombination

Homologous recombination is a source of diversity in both natural and directed evolution. Standing genetic variation that has passed the test of natural selection is combined in new ways, generating functional and sometimes unexpected changes. In this work we evaluate the utility of homologous recombination as a protein engineering tool, both in comparison with and combined with other protein engineering techniques, and apply it to an industrially important enzyme: Hypocrea jecorina Cel5a.

Chapter 1 reviews work over the last five years on protein engineering by recombination. Chapter 2 describes the recombination of Hypocrea jecorina Cel5a endoglucanase with homologous enzymes in order to improve its activity at high temperatures. A chimeric Cel5a that is 10.1 °C more stable than wild-type and hydrolyzes 25% more cellulose at elevated temperatures is reported. Chapter 3 describes an investigation into the synergy of thermostable cellulases that have been engineered by recombination and other methods. An engineered endoglucanase and two engineered cellobiohydrolases synergistically hydrolyzed cellulose at high temperatures, releasing over 200% more reducing sugars over 60 h at their optimal mixture relative to the best mixture of wild-type enzymes. These results provide a framework for engineering cellulolytic enzyme mixtures for the industrial conditions of high temperatures and long incubation times.

In addition to this work on recombination, we explored three other problems in protein engineering. Chapter 4 describes an investigation into replacing enzymes with complex cofactors with simple cofactors, using an E. coli enolase as a model system. Chapter 5 describes engineering broad-spectrum aldehyde resistance in Saccharomyces cerevisiae by evolving an alcohol dehydrogenase simultaneously for activity and promiscuity. Chapter 6 describes an attempt to engineer gene-targeted hypermutagenesis into E. coli to facilitate continuous in vivo selection systems.

Structurally engineered cytochromes c with novel ligand-binding properties

Semisynthesis of horse heart cytochrome c and site-directed mutagenesis of Saccharomyces cerevisiae (S. c.) iso-1-cytochrome c have been utilized to substitute Ala for the cytochrome c heme axial ligand Met80 to yield ligand-binding proteins (horse heart Ala80cyt c and S.c. Ala80cyt c) with spectroscopic properties remarkably similar to those of myoglobin. Both species of Fe(II)Ala80cyt c form exceptionally stable dioxygen complexes with autoxidation rates 10-30x smaller and O₂ binding constants ~ 3x greater than those of myoglobin. The resistance of O₂-Fe(II)Ala80cyt c to autoxidation is attributed in part to protection of the heme site from solvent as exhibited by the exceptionally slow rate of CO binding to the heme as well as the low quantum yield of CO photodissociation.

UV/vis, EPR, and paramagnetic NMR spectroscopy indicate that at pH 7 the Fe(III)Ala80cyt c heme is low-spin with axial His-OH^- coordination and that below pH ~6.5, Fe(III)Ala80cyt cis high-spin with His-H₂O heme ligation. Significant differences in the pH dependence of the ¹H NMR spectra of S.c. Fe(III)Ala80cyt c compared to wild-type demonstrate that the axial ligands influence the conformational energetics of cytochrome c.

¹H NMR spectroscopy has been utilized to determine the solution structure of the cyanide derivative of S.c. Fe(III)Ala80cyt c. 82% of the resonances in the ¹H NMR spectrum of S.c. CN-Fe(III)Ala80cyt c have been assigned through 1D and 2D experiments. The RMSD values after restrained energy minimization of the family of 17 structures obtained from distance geometry calculations are 0.68 ± 0.11 Å for the backbone and 1.32 ± 0.14 Å for all heavy atoms. The solution structure indicates that a tyrosine in the "distal" pocket of CN-Fe(III)Ala80cyt c forms a hydrogen bond with the Fe(III)-CN unit, suggesting that it may play a role analogous to that of the distal histidine in myoglobin in stabilizing the dioxygen adduct.

Developmentally regulated transcription factors in Drosophila melanogaster

During early stages of Drosophila development the heat shock response cannot be induced. It is reasoned that the adverse effects on cell cycle and cell growth brought about by Hsp70 induction must outweigh the beneficial aspects of Hsp70 induction in the early embryo. Although the Drosophila heat shock transcription factor (dHSF) is abundant in the early embryo, it does not enter the nucleus in response to heat shock. In older embryos and in cultured cells the factor is localized within the nucleus in an apparent trimeric structure that binds DNA with high affinity. The domain responsible for nuclear localization upon stress resides between residues 390 and 420 of the dHSF. Using that domain as bait in a yeast two-hybrid system we now report the identification and cloning of a nuclear transport protein Drosophila karyopherin-α3(dKap- α3). Biochemical methods demonstrate that the dKap-α3 protein binds specifically to the dHSF's nuclear localization sequence (NLS). Furthermore, the dKap-α3 protein does not associate with NLSs that contain point mutations which are not transported in vivo. Nuclear docking studies also demonstrate specific nuclear targeting of the NLS substrate by dKap-α3.Consistant with previous studies demonstrating that early Drosophila embryos are refractory to heat shock as a result of dHSF nuclear exclusion, we demonstrate that the early embryo is deficient in dKap-α3 protein through cycle 12. From cycle 13 onward the transport factor is present and the dHSF is localized within the nucleus thus allowing the embryo to respond to heat shock.

The pair-rule gene fushi tarazu (ftz) is a well-studied zygotic segmentation gene that is necessary for the development of the even-numbered parasegments in Drosophila melanogastor. During early embryogenesis, ftz is expressed in a characteristic pattern of seven stripes, one in each of the even-numbered parasegments. With a view to understand how ftz is transcriptionally regulated, cDNAs that encode transcription factors that bind to the zebra element of the ftz promoter have been cloned. Chapter Ill reports the cloning and characterization of the eDNA encoding zeb-1 (zebra element binding protein), a novel steroid receptor-like molecule that specifically binds to a key regulatory element of the ftz promoter. In transient transfection assays employing Drosophila tissue culture cells, it has been shown that zeb-1 as well as a truncated zeb-1 polypeptide (zeb480) that lacks the putative ligand binding domain function as sequencespecific trans-activators of the ftz gene.

The Oct factors are members of the POU family of transcription factors that are shown to play important roles during development in mammals. Chapter IV reports the eDNA cloning and expression of a Drosophila Oct transcription factor. Whole mount in-situ hybridization experiments revealed that the spatial expression patterns of this gene during embryonic development have not yet been observed for any other gene. In early embryogenesis, its transcripts are transiently expressed as a wide uniform band from 20-40% of the egg length, very similar to that of gap genes. This pattern progressively resolves into a series of narrower stripes followed by expression in fourteen stripes. Subsequently, transcripts from this gene are expressed in the central nervous system and the brain. When expressed in the yeast Saccharomyces cerevisiae, this Drosophila factor functions as a strong, octamer-dependent activator of transcription. The data strongly suggest possible functions for the Oct factor in pattern formation in Drosophila that might transcend the boundaries of genetically defined segmentation genes.

Studies of dinucleoside monophosphates and monomer-polynucleotide interactions by proton magnetic resonance

The nature of the intra- and intermolecular base-stacking interactions involving several dinucleoside monophosphates in aqueous solution have been investigated by proton magnetic resonance spectrosocopy, and this method has been applied to a study of the interaction of polyuridylic acid with purine and adenosine monomers.

The pmr spectra of adenylyl (3' → 5') cytidine (ApC) and cytidylyl (3' → 5') adenosine (CpA) have been studied as a function of concentration and temperature. The results of these studies indicate that the intramolecular base-stacking interactions between the adenine and cytosine bases of these dinucleoside monophosphates are rather strong, and that the stacking tendencies are comparable for the two sequence isomers. The chemical shifts of the cytosine H₅ and adenine H₂ protons, and their variations with temperature, were shown to be consistent with stacked conformations in which both bases of the dinucleoside monophosphates are preferentially oriented in the anti conformation as in similar dApdC, and dCpdA (dA = deoxyadenosine; dC = deoxycytidine) segments in double helical DNA. The intramolecular stacking interaction was found to have a pronounced effect on the conformations of the ribose moieties, and these conformational changes are discussed. The concentration studies indicate extensive self-association of these dinucleoside monophosphates, and analysis of the concentration data facilitated determination of the dimerization constant for the association process as well as the nature of the intermolecular complexes.

The dependence of the ribose conformation upon the extent of intramolecular base-stacking was used to demonstrate that the base-base interaction in cytidylyl (3' → 5') cytidine (CpC) is rather strong, while there appears to be little interaction between the two uracil bases of uridylyl (3' → 5') uridine (UpU).

Studies of the binding of purine to several ribose and deoxyribose dinucleoside monophosphates show that the mode of interaction is base-stacking, and evidence for the formation of a purine-dinucleoside monophosphate intercalated complex is presented. The purine proton resonances are markedly broadened in this complex, and estimates of the purine linewidths in the complex and the equilibrium constant for purine intercalation are obtained.

A study of the interaction of unsubstitued purine with polyuridylic acid at 29°C by pmr indicated that purine binds to the uracil bases of the polymer by base-stacking. The severe broadening of the purine proton resonances observed provides strong evidence for the intercalation of purine between adjacent uracil bases of poly U. This interaction does not result in a more rigid or ordered structure for the polymer.

Investigation of the interaction between adenosine and polyuridylic acid revealed two modes of interaction between the monomer and the polymer, depending on the temperature. At temperatures above 26°C or so, monomeric adenosine binds to poly U by noncooperative A-U base stacking. Below this temperature, a rigid triple-stranded 1A:2U complex is formed, presumably via cooperative hydrogen-bonding as has previously been reported.

These results clearly illustrate the importance of base-stacking in non-specific interactions between bases, nucleosides and nucleotides, and also reveal the important role of the base-stacking interactions in cooperatively for med structures involving specific base-pairing where both types of interaction are possible.

Isolation and characteristics of a bacteriophage for bacillus stearothermophilus

A bacteriophage (TØ3) which infects the thermophilic bacterium Bacillus stearothermophilus ATCC 8005 was isolated and characterized. Infection of the bacterium by the bacteriophage was carried out at 60°C, the optimum growth temperature of the host. At 60°C the phage has a latent period of 18 minutes and a burst size of about 200. The phage is comparatively thermostable in broth. The half life of the phage is 400 minutes at 60°C, 120 minutes at 65°C, 40 minutes at 70°C and 12 minutes at 75°C. The activation energy for the heat inactivation of TØ3 is 56,000 cal. The buoyant density of TØ3 in a cesium chloride density gradient is 1.526.

Electron micrographs of TØ3 indicate that the phage has a regular hexagonal shaped head 57 mμ long. The morphology of the head is compatible with icosahedral symmetry. Each edge of the head is 29 mμ long, and there are 6 or 7 subunits along each edge. The tail of TØ3 is 125 mμ long and 10 mμ wide. There are about 30 cross striations that are spaced at 3.9 mμ intervals along the tail.

The DNA of phage TØ3 has a melting temperature of 88.5°C. Heat denatured TØ3 DNA can be extensively annealed in a high ionic strength environment. The buoyant density of TØ3 DNA in a cesium chloride density gradient is 1.695. TØ3 DNA contains: 42.7% guanine plus cytosine, as determined from the melting temperature; 43% guanine plus cytosine, as determined from the buoyant density; and 40.2% guanine plus cytosine, as determined by chromatographic separation and spectrophotometric estimation of the bases. The molecular weight of TØ3 DNA is 16.7 X 10⁶ as determined from the band width of the TØ3 DNA concentration distribution in a cesium chloride density gradient. Electron microscopy of TØ3 DNA revealed a single linear molecule that is 11.7 μ long. This corresponds to a molecular weight of 22.5 X 10⁶.

Heat denatured TØ3 DNA forms two bands in a cesium chloride density gradient, one at a density of 1.707 and the other at a density of 1.715. After the separated bands are mixed and annealed in the centrifuge cell, the renatured TØ3 DNA forms a single band at a density of 1.699. These results indicate that the two complementary strands of TØ3 DNA have different buoyant densities in cesium chloride, presumably because they have different base compositions.

The characteristics of TØ3 are compared with those of other phages. A hypothesis is presented for a relationship between the base composition of one strand of TØ3 DNA and the amino acid composition of the proteins of TØ3.

I. Proton magnetic resonance of polynucleotides and transfer RNA. II. Electron spin relaxation studies of manganese (II) complexes in acetonitrile

Part I. Proton Magnetic Resonance of Polynucleotides and Transfer RNA.

Proton magnetic resonance was used to follow the temperature dependent intramolecular stacking of the bases in the polynucleotides of adenine and cytosine. Analysis of the results on the basis of a two state stacked-unstacked model yielded values of -4.5 kcal/mole and -9.5 kcal/mole for the enthalpies of stacking in polyadenylic and polycytidylic acid, respectively.

The interaction of purine with these molecules was also studied by pmr. Analysis of these results and the comparison of the thermal unstacking of polynucleotides and short chain nucleotides indicates that the bases contained in stacks within the long chain poly nucleotides are, on the average, closer together than the bases contained in stacks in the short chain nucleotides.

Temperature and purine studies were also carried out with an aqueous solution of formylmethionine transfer ribonucleic acid. Comparison of these results with the results of similar experiments with the homopolynucleotides of adenine, cytosine and uracil indicate that the purine is probably intercalating into loop regions of the molecule.

The solvent denaturation of phenylalanine transfer ribonucleic acid was followed by pmr. In a solvent mixture containing 83 volume per cent dimethylsulf oxide and 17 per cent deuterium oxide, the tRNA molecule is rendered quite flexible. It is possible to resolve resonances of protons on the common bases and on certain modified bases.

Part II. Electron Spin Relaxation Studies of Manganese (II) Complexes in Acetonitrile.

The electron paramagnetic resonance spectra of three Mn⁺² complexes, [Mn(CH₃CN)₆]⁺², [MnCl₄]^-2, and [MnBr₄]^-2, in acetonitrile were studied in detail. The objective of this study was to relate changes in the effective spin Hamiltonian parameters and the resonance line widths to the structure of these molecular complexes as well as to dynamical processes in solution.

Of the three systems studied, the results obtained from the [Mn(CH₃CN)₆]⁺² system were the most straight-forward to interpret. Resonance broadening attributable to manganese spin-spin dipolar interactions was observed as the manganese concentration was increased.

In the [MnCl₄]^-2 system, solvent fluctuations and dynamical ion-pairing appear to be significant in determining electron spin relaxation.

In the [MnBr₄]^-2 system, solvent fluctuations, ion-pairing, and Br- ligand exchange provide the principal means of electron spin relaxation. It was also found that the spin relaxation in this system is dependent upon the field strength and is directly related to the manganese concentration. A relaxation theory based on a two state collisional model was developed to account for the observed behavior.