Author

Zhong Wang

Date

August 2000

Document Type

Dissertation

Degree Name

Ph.D.

Department

Dept. of Biochemistry and Molecular Biology

Institution

Oregon Graduate Institute of Science & Technology

Abstract

The major focus of this study is to elucidate the mechanism of translational control of arg-2. arg-2 is a gene involved in Neurospora crassa arginine (Arg) biosynthesis, and translation of ARG2 is negatively regulated by the availability of Arg. This regulation is mediated by a short upstream open reading frame (uORF) in the arg-2 transcript. The mechanism for this specific regulation has been addressed: (i) We developed an amino acid-dependent cell-free translation system in which the Arg-specific regulation is reconstituted. (ii) We introduced a sensitive assay called "toeprinting" (primer extension inhibition) into this cell-free system. We showed that, in high Arg, the translation of the arg-2 uORF-encoded Arg attenuator peptide (AAP) causes ribosomes to stall precisely at the position in which the uORF termination codon is in the ribosomal A site. Point mutations which changed single amino acids within the conserved AAP region abolish this Arg-specific ribosomal stalling. (iii) We elucidated the role of the AAP by directly fusing it to the N-terminus of a longer polypeptide. In high Arg, elongating ribosomes are stalled just after they have translated the AAP region. Thus, the AAP appears to function as a nascent peptide that acts in cis to cause regulated stalling of ribosomes. (iv) We investigated the generality of AAP-mediated regulation. Both the N. crassa arg-2 and Saccharomyces cerevisiae CPAl AAP retain their regulatory effects in N. crassa, S. cerevisiae, and wheat germ extracts. These studies further revealed that AAP-mediated Arg-specific regulation appears independent of the charging status of arginyl-tRNAs. This work represents the first instance in which translational control in response to the availability of a single amino acid has been reconstituted in a eukaryotic cell-free translation system. It represents one of a handful of examples of how a uORF-encoded peptide controls protein synthesis is understood. Many of the genes involved in growth control, development, and cancer encode such short peptides in uORFs. Understanding how the fungal AAPs mediate Arg-specific regulation provides key insights into fundamental aspects of translation and helps advance our understanding of how such uORFs mediate translational control.

Identifier

doi:10.6083/M4542KJQ

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