Author

Cheng Wu

Date

March 2008

Document Type

Dissertation

Degree Name

Ph.D.

Department

Dept. of Environmental and Biomolecular Systems

Institution

Oregon Health & Science University

Abstract

The Neurospora crassa arg-2 gene encodes the small subunit of carbamoylphosphate synthetase, the first enzyme in fungal arginine (Arg) biosynthesis, which is negatively feed-back regulated by the cellular arginine level. This regulation is mediated by a 24-residue peptide, known as the arginine attenuator peptide (AAP),specified by an evolutionarily conserved upstream open reading frame (uORF) in arg-2m RNA. Previous studies with cell-free translation systems from N. crassa and Saccharomyces cerevisiae have shown that ribosomes synthesizing the N. crassa arg-2 and the homologous S. cerevisiae CPA1 uORF-encoded AAPs stall at the uORF termination codon in the presence of high levels of Arg, blocking ribosomes froms canning to the downstream initiation codon. The use of cell-free translation systems from fungi and primer extension inhibition assays has provided key insights into the mechanism underlying the AAP-mediated ribosome stalling. Here, I present results suggesting that (i) the nascent AAP can transiently stall ribosomes when placed either at the N-terminus or internally within a polypeptide; (ii) ribosomes appear to resume translation after release from the stalling; and (iii) the nascent AAP is in its peptidyl-tRNA form while protruding into the ribosome exit tunnel when the ribosome stalls after synthesizing the AAP. The 18-residue 2A peptide of foot-and-mouth disease virus (FMDV) mediates aco-translational cleavage at its C-terminus resulting in separated N- and C-terminal products. The 2A reaction was recapitulated in the N. crassa cell-free translation systems. The majority of N-terminal products are released peptides when the ribosome pause occurs instead of being linked to the tRNA. Primer extension inhibition assays revealed a ribosome pause at the C-terminus of 2A. These results suggest that the nascent 2A peptide adopts a confirmation that stalls the translating ribosome and promotes the hydrolysis of the peptidyl-tRNA bond. Salmonella enterica alternatively expresses two distinct types of flagellin proteins, FljB and FliC. FljA is co-expressed with FljB to inhibit FliC expression. Mutations that allow synthesis of FliC in the presence of FljA are located in the 5'-UTR of the fliC mRNA, which suggests that FljA binds to this region to inhibit translation. A modified primer extension inhibition assay was used to map the position of bound FljA on fliC mRNA, revealing that the binding site overlaps the ribosome binding site. When FljA binds to the fliC mRNA, it blocks ribosomes access to the translation initiation site,thereby inhibiting the synthesis of FliC. The successful use of cell-free translation systems and primer extension inhibition assays in studying these three cases proves they are powerful tools that enable biochemical determination of factors and mechanisms contributing to translational processes.

Identifier

doi:10.6083/M4FN1447

School

OGI School of Science and Engineering

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