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Journal of Undergraduate Research

Keywords

peptides, ribosome stalling, 50S, 30S, protein synthesis

College

Physical and Mathematical Sciences

Department

Chemistry and Biochemistry

Abstract

The ribosome is a macromolecular machine found in all living cells. It catalyzes protein synthesis through a process known as translation. The ribosome reads messenger RNA that carries the blueprint for a protein product and brings in the appropriate amino acids to build a specific protein. Ribosomes are composed of two subunits (50S and 30S) that form three binding sites for transfer RNA. The A site of the ribosome accepts the incoming tRNA charged with an amino acid. This tRNA transfers its amino acid to a growing polypeptide chain held in the P site. After the transfer, the tRNA exits the ribosome through the E site. Previous studies revealed that certain nascent–newly forming–peptide chains stall ribosomes during protein synthesis. Stalling has been shown to play a significant role in gene regulation. For example, SecM is a peptide that stalls the ribosome when there are low levels of the bacterial-motor protein SecA. This stalling event allows the ribosome to synthesize more SecA protein. Previous studies have shown that only a few amino acids are required for stalling. These known stalling peptide sequences are short and simple but lack similarity and show tolerance to mutation. This suggests that there may be undiscovered stalling motifs within other E. coli protein. We used a new genetic selection, called the two-hybrid system, to find additional stalling sequences from a collection of random sequences (a library). This library consisted of 108 sequences, each having twenty random amino acids. We found several novel stalling sequences, and my responsibility was to locate the exact site of stalling in the ribosome. I performed deletions to isolate the minimal sequence motif needed for stalling and performed mutagenesis studies to find which amino acids are critical for stalling the ribosome. From this data, we expected to formulate rules as to what is necessary and sufficient for stalling. These rules we could then be used in the future to identify endogenous proteins where stalling may play a role in gene expression or protein folding.

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