Microbial degradation of plant biomass plays a central role in the global carbon cycle. However, the turnover of cellulose in plants poses a difficult challenge due to its microcrystalline nature and its protection by other polymers in the plant cell wall. Bacteria and fungi have evolved diverse strategies for breaking down plant biomass, involving suites of cellulolytic enzymes that are produced at appropriate times to degrade the recalcitrant substrate. The work presented here provides an analysis of two cellulolytic organisms and their potential as model systems in the study of cellulose degradation.

We probed the cellulolytic system in Clostridium papyrosolvens C7 by sequencing its genome and characterizing the cellulosomal components. Comparisons to other cellulosome producing organisms revealed many unique enzymes and non-catalytic cellulosome components. When grown on different carbon sources, C. papyrosolsovens C7 upregulates enzymes incorporated into cellulosomes with surprisingly little compositional diversity or dynamic range. These results provide a framework for using C. papyrosolvens C7 in the engineering of designer cellulosomes, as well as understanding the regulation and efficiency of these heterogeneous multienzyme complexes.

The tools available for studying N. crassa have recently been applied to its cellulolytic system and much has been quickly learned about its degradative capability. However, the processes that produce its strong and specific induction of cellulase genes are not well understood. Through a screen for deletion strains that affect cellulose degradation, a kinase (NCU07399) was identified that seems to affect the transcriptional activation of several cellulase genes. Preliminary characterization of this kinase reveals an unusual domain architecture and in vitro phosphorylation activity that mimic studies on its yeast and human homologs. The screen and subsequent assays used to analyze NCU07399 presented here should provide a basis for dissecting the pathways involved in cellulase upregulation by N. crassa.

Improper regulation of translation initiation, a vital check-point of protein synthesis in the cell, has been linked to a number of cancers. Overexpression of protein subunits of eukaryotic translation initiation factor 3 (eIF3) has been associated with increased translation of mRNAs involved in cell proliferation. In addition to playing a major role in general translation initiation by serving as a scaffold for the assembly of translation initiation complexes, eIF3 regulates translation of specific cellular mRNAs and viral RNAs. Mutations in the N-terminal Helix-Loop-Helix (HLH) RNA-binding motif of the EIF3A subunit in eIF3 interfere with Hepatitis C Virus Internal Ribosome Entry Site (IRES) mediated translation initiation in vitro. Here we show that the EIF3A HLH motif controls translation of a small set of cellular transcripts enriched in oncogenic mRNAs, including MYC. We also demonstrate that the HLH motif of EIF3A acts specifically on the 5’-UTR of MYC mRNA and modulates the function of EIF4A1 on select transcripts during translation initiation. In Ramos lymphoma cell lines, which are dependent on MYC overexpression, mutations in the HLH motif greatly reduce MYC expression, impede proliferation and sensitize cells to anti-cancer compounds. These results reveal the potential of the EIF3A HLH motif in eIF3 as a promising chemotherapeutic target.

The ribosome catalyzes the synthesis of polypeptides with high efficiency and sequence specificity. Repurposing the ribosome as a platform for manufacturing other sequence defined polymers could access a wide variety of previously unattainable molecules and bulk materials. In this work, we aim to understand both engineered and wild type ribosomes through structural and biochemical analysis. We reveal limitations of previous engineering efforts on the ribosome, highlighting the importance of careful mutation and selection techniques. The ribosome we study is poorly assembled and nonfunctional in vitro despite improved polymerization of β-amino acids in vivo. We identify key regions of the ribosome that are disrupted by mutations and offer suggestions for more targeted engineering that will preserve efficient ribosome assembly. We also characterize the structure of the wild type ribosome bound to an unnatural, non-α-amino acid monomer for the first time. This monomer is correctly accommodated into the P site of the ribosome, explaining previously observed activity as an initiator substrate. Lastly, we assess a new, short peptide luciferase-complementing reporter in defined in vitro translations as a better readout of mutant ribosome activity. Using this assay, we show activity of purified active site mutant ribosomes in vitro for the first time, reconciling often observed differences from in vivo systems. These projects underscore the importance of detailed characterization of both the input to and results of ambitious bioengineering efforts.

mRNA translation regulation by eukaryotic initiation factors (eIFs) is crucial for cell survival. In humans, eIF3 stimulates translation of the JUN mRNA which encodes the transcription factor JUN, an oncogenic transcription factor that is involved in cell cycle progression, apoptosis, and cell proliferation. Previous studies revealed that eIF3 activates translation of the JUN mRNA by interacting with a stem loop in the 5′ untranslated region (5′ UTR) and with the 5′ -7-methylguanosine cap structure. In addition to its interaction site with eIF3, the JUN 5′ UTR has a longer than average length, a high degree of secondary structure, high GC content, and an upstream start codon (uAUG). This motivated us to explore the complexity of JUN mRNA translation regulation in human cells. Chapter 2 describes our findings on the contributions of multiple 5’ UTR and start codon features in JUN translation regulation. We find that JUN translation is regulated in a sequence and structure-dependent manner in regions adjacent to the eIF3-interacting site in the JUN 5′ UTR. Furthermore, we identify contributions of an additional initiation factor, eIF4A, in JUN regulation. We show that enhancing the interaction of eIF4A with JUN by using the compound Rocaglamide A (RocA) represses JUN translation. We also find that both the upstream AUG (uAUG) and the main AUG (mAUG) contribute to JUN translation and that they are conserved throughout vertebrates. Work presented in this chapter demonstrates additional layers of regulation for JUN translation. Chapter 3 describes isolation of a translation initiation multifactor complex (MFC) from in vitro translation reactions of a JUN 5’UTR reporter mRNA in human cell extracts. The yeast MFC is composed of eIF1, eIF2, eIF3, eIF5 and the initiator methionyl-tRNA (Met-tRNAi) and has been well-characterized. However, knowledge about the human MFC is limited and isolation of the endogenous complex from human cells hasn’t been achieved. By using the JUN mRNA as a platform for MFC binding, we present the first instance of isolation of an mRNA-bound human MFC. We also present in-depth protocol optimization and propose next steps for complex validation. Work presented in this chapter provides strong evidence for the formation of a human MFC with the presence of novel initiation factors such as eIF4A and eIF4G. Together these findings demonstrate the complexity of JUN translation regulation and establish JUN’s potential as a model transcript for understanding multiple interacting modes of translation regulation.

Historically, fermentation has allowed the production of a myriad of food products that acquired central roles in human society such as bread, beer and wine. Through the centuries, fermentation has grown into a billion dollar industry; gaining traction for the production of pharmaceuticals, renewable plastics and new materials. The yeast Saccharomyces cerevisiae ("baker's yeast"), continues to be used by most industries; however, novel species are necessary to attend the growing and diversifying demand as S. cerevisiae has proven difficult to engineer to expand the carbon sources it can use, the products it can make, and the harsh conditions it can tolerate in industrial applications. Other yeast species, which frequently contain inherent features addressing many of these issues, remain difficult to manipulate genetically. Here, we engineer one of the most promising candidates - the thermotolerant yeast Kluyveromyces marxianus - to create a new synthetic biology platform. Using CRISPR-Cas9 mediated genome editing, we show that wild isolates of K. marxianus can be made heterothallic for sexual crossing. By breeding two of these mating-type engineered K. marxianus strains, we combined three complex traits - thermotolerance, lipid production, and facile transformation with exogenous DNA - into a single host. The ability to cross K. marxianus strains with relative ease, together with CRISPR-Cas9 genome editing, should enable engineering of K. marxianus isolates with promising lipid production at temperatures far exceeding those of other fungi under development for industrial applications. These results establish K. marxianus as a synthetic biology platform comparable to S. cerevisiae, with naturally more robust traits that hold potential for the industrial production of renewable chemicals. K. marxianus was also successfully employed as a model organism; providing a new platform for a comprehensive, mechanistic, large-scale study of innate thermotolerance. From a library of mutants to identify genes related to innate thermotolerance, we identified 7 mutants that showed impaired growth at high temperature, indicating a potential role of these genes in the high temperature growth phenotype. This work provides a starting point to elucidate the mechanistic basis underlying the ability to sustain growth at high temperatures, the basis of which is still largely unknown in eukaryotes.

Liquid fuels derived from plant biomass have the potential to contribute to sustainability efforts. However, the conversion of plant biomass is very difficult due to its complex nature. Cellulose, the primary component of plant cell walls, is a recalcitrant polysaccharide comprised of glucose. In nature, various fungi and bacteria enzymatically depolymerize cellulose for consumption. Understanding how cellulases are produced and function will be invaluable to the realization of cellulosic biofuels. The work here describes how a prominent cellulase interacts with cellulose during depolymerization, and how the cellulolytic fungus Neurospora crassa in part signals for the production of cellulase enzymes.

The interactions of cellulases with cellulose are not well understood due to the recalcitrance and heterogeneity of the solid substrate. Cellobiohydrolase I, the most prominent cellulase in many fungal systems, was fluorescently labeled and monitored on the surface of crystalline cellulose. Four classes of behaviors were observed for this enzyme on the surface of cellulose: productive and linear movement, nonproductive surface diffusion, stationary, and transient association. In parallel, changes to the surface of crystalline cellulose were observed with atomic force microscopy to characterize how this enzyme modifies its substrate after degradation.

The processes that initiate cellulase induction in Neurospora crassa have yet to be identified. A screen of deletion strains grown on cellulose was performed to isolate regulators of cellulase production. A kinase (NCU07399) was identified as a regulator of the transcriptional activation of several cellulase genes. Deletion of this kinase in Neurosporareduces the upregulation of cellulases by as much as 50-fold and also induces the secretion of a metalloprotease. The results here provide a basis for exploring the pathways involved in N. crassa cellulase production.

Protein synthesis takes place in four stages: initiation, elongation, termination and recycling. Elongation consists of delivery of a charged tRNA to the ribosome, peptide bond formation and translocation of the mRNA and tRNA, three steps forming a cyclic process, which is repeated for every amino acid added to a growing polypeptide chain. The result of the elongation cycle is the translation of the triplet genetic code contained in an mRNA, into the amino acid sequence of a protein. Since the 1960s it was appreciated that complex conformational changes must occur on the ribosome to accomplish translocation of mRNA and tRNA. Here I report x-ray crystallographic studies which shed light on how mRNA and tRNA are manipulated by the ribosome and mechanisms used by certain antibiotics to inhibit the elongation cycle of protein synthesis.

Specifically research reported here argue that tRNA translocation is a stepwise process that involves discrete structural intermediates of the ribosome. I report structural evidence that the antibiotics clindamycin and chloramphenicol inhibit protein synthesis by interfering with aminoacyl-tRNA positioning in the peptidyl transferase center. Also, I hypothesize based on structural data and phylogenetic analysis, that the identity of the ribosomal RNA residues numbered in E. coli 752, 2055 and 2609 contribute to the specificity of many antibiotics for binding to bacterial, rather than archaeal or eukaryotic ribosomes.