Author

Peng Fang

Date

August 2003

Document Type

Dissertation

Degree Name

Ph.D.

Department

Dept. of Biochemistry and Molecular Biology

Institution

Oregon Health & Science University

Abstract

The arginine attenuator peptides (AAP), specified by an evolutionarily conserved upstream open reading frame (uORF) in mRNAs specifying small subunit of carbamoyl phosphate synthetase in fungi, can control the movement of ribosomes in response to arginine. Previous studies with cell-free translation systems from Neurospora crassa and Saccharomyces cerevisiae have indicated that ribosomes synthesizing the N. crassa arg-2 and the homologous S. cerevisiae CPAI uORF-encoded AAPs stall at the uORF termination codon when the concentration of Arg is high, blocking ribosomes from scanning to the downstream initiation codon. These AAPs also cause Arg-regulated stalling of ribosomes involved in elongation when fused at the N-terminus of a luciferase (LUC) reporter. The research work for this dissertation is focused on elucidating the detailed mechanism of the AAP-mediated translational control in vitro with cell-free systems, which includes analyzing cis-acting requirements for arg-2 uORF function, examining effects of existing trans-acting mutations that cause translation defects in vivo on arg-2 regulation, and investigating the regulated ribosome movement by a nascent AAP domain within a larger polypeptide. The results indicate that: (i) The conserved AAP sequence, but not the mRNA sequence, appears responsible for regulation. The highly evolutionarily conserved core of the peptide functions within the ribosome to cause stalling. Translational events at a potential stall site, such as an encounter with a stop codon or a rare codon, can influence the extent of stalling at that site. (ii) Seven tested N crassa super-suppressor strains (ssu- 1, -2, -3, -4, - 5, -9, and -10) produce amber suppressor-tRNA, but these mutant strains appear unaltered in arginine regulation. (iii) The AAP domain can transiently stall ribosomes when placed either at the N-terminus or within a polypeptide. The half-life of ribosomes stalled by the AAP during elongation increased when the Arg concentration increased. Ribosomes appear to resume translation after release from the stall. This work demonstrates that a regulatory uORF-encoded peptide mediates translational control as a nascent peptide within the ribosome.

Identifier

doi:10.6083/M4DV1GT3

School

OGI School of Science and Engineering

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