Mutational Analysis of Transcriptional Initiation in Bacteria

pharmaceuticals, attack cancer cells, neutralize environmental pollutants, and synthesize biofuels (Khalil & Collins, 2010). Gene expression begins with transcription, the process by which DNA information, in the form of the base sequence of a gene, is converted into RNA base sequence information. For genes that encode proteins, the RNA product of transcription is used during translation to encode the sequence of amino acids in a protein. As the first step in gene expression, transcription is an important control point for gene regulation. Initiation of transcription in bacteria involves binding of an enzyme called RNA polymerase to a sequence of DNA called a transcriptional promoter. As illustrated in Figure 1, a common form of bacterial promoters includes two conserved sequence elements, a -35 region that is recognized during transcriptional initiation by RNA polymerase and its associated Sigma factor, and a -10 region that is involved in DNA melting (Ross, Aiyar, Salomon, & Gourse, 1998). The consensus sequence for the -35 region of E. coli promoters has been widely reported to be TTGACA (Harley & Reynolds, 1987; Lisser & Margalit, 1993). The consensus sequence of the -10 region is TATAAT (Waterman, Arratia, & Galas, 1984). The RNA polymerase attaches itself to one of the two DNA strands referred to as the template strand and begins to use it to make RNA. The RNA polymerase proceeds to slide along the template strand for the entire length of the gene, reading it in a 3’ to 5’ direction. Transcription ceases when the RNA polymerase encounters a transcriptional terminator. In bacteria, the resulting RNA transcript is used for translation as soon as it is available. Mutational analysis of the -35 region of naturally occurring promoters showed that the -35 region is involved in the initial binding of the RNA polymerase to the promoter, and that it is an important contributor to the overall strength of a bacterial promoter. An in vitro study of the effect of mutations in the -35 region on the INTRODUCTION Gene expression is the process by which gene information is used to direct the function of cells. It is regulated in all cells because not all genes are required all the time or under all circumstances. For example, human brain cells need to express certain genes that are not needed in muscle cells, and vice versa (Gurdon & Melton, 2008). In a similar sense, bacteria must express different genes depending on temperature, pH, osmotic pressure, or the availability of food (Beales, 2004). Knowledge of gene regulation is important for understanding the differentiation and function of eukaryotic cells, the development of tissues in multicellular organisms, and the relationships of bacteria to their environments throughout the biosphere. It helps us to understand genetic diseases, diseases impacted by genetics, and cellular disorders such as cancer (LópezBigas & Ouzounis, 2004). Gene regulation research can also be used to explore the contribution of bacterial microbiomes to human health and disease (Cho & Blaser, 2012). In the context of synthetic biology, research results on the regulation of gene expression are being applied to engineer bacterial cells that produce Mutational Analysis of Transcriptional Initiation in Bacteria