rClone: a synthetic biology tool that enables the research of bacterial translation

Abstract

mRNA, called the ribosome binding site (RBS; Figure 1). After the small ribosomal subunit binds to the RBS, the large ribosomal subunit attaches to the small subunit to begin translation of the mRNA into a chain of amino acids. The mRNA bases are read as triplet codons that interact by base pairing with anticodons in transfer RNA (tRNA) molecules, which carry amino acids to the growing protein chain (Malys & McCarthy, 2010). As shown in Figure 1, RNA-RNA base pairing typically involves the Watson-Crick base pairs of G with C, and A with U, but G can also base pair with U. The conventional understanding is that the strength of a given RBS is determined by the strength of its base pairing interactions with the 16S rRNA (Shine & Dalgarno, 1974). In natural bacterial genomes, there is a wide variety of RBS sequences and RBS translational strengths that have resulted from natural selection for global patterns of gene expression. The relationship between RNA base pairing and the strength of an RBS also explains how synthetic RBSs can be produced with widely varying strengths. In addition to intermolecular base pairing, intramolecular base pairing affects the strengths of RBSs. The ability of RNA to engage in intramolecular base pairing is well established (Busan & Weeks, 2013). RBS elements can be disabled by intramolecular RNA folding, as is the case in riboswitches (Breaker, 2012). The RNA in riboswitches adopts an OFF state when the RBS is bound by a complementary anti-RBS sequence within the mRNA. For the ON state, a small molecule ligand binds to the folded RNA and changes the RNA shape so that the RBS is available for interaction with the 16S rRNA. Understanding the function of RBSs informs the discipline of synthetic biology, which uses engineering principles and molecular cloning methods for the construction of parts, devices, and systems, INTRODUCTION Gene expression, the process by which the inherited information of genes is used to direct the function of cells, is regulated in all cells because not all genes are needed all the time or under all circumstances (Hijum, Medema, & Kuipers, 2009). Gene expression begins with transcription, the process by which the DNA base sequence of a gene is converted into RNA sequence information. For genes that encode proteins, the messenger RNA (mRNA) product of transcription is used during translation to encode the sequence of amino acids in a protein. The sequence of bases in mRNA is translated by the ribosome, which is composed of a large (50S) and a small (30S) subunit. Translation is initiated when the 16S ribosomal RNA (rRNA) of the small ribosomal subunit base pairs to a conserved sequence in the rClone: A Synthetic Biology Tool That Enables the Research of Bacterial Translation