Читать книгу Snyder and Champness Molecular Genetics of Bacteria - Tina M. Henkin - Страница 243

The concept of an open reading frame, or ORF, is very important, particularly in this age of genomics. As discussed above, a reading frame in DNA is a succession of nucleotides in the DNA taken three at a time, the same way the genetic code is translated. Each DNA sequence has six reading frames, three on each strand, as illustrated in Figure 2.44. An ORF is a string of potential codons for amino acids in DNA unbroken by termination codons in one of the reading frames. Computer software can show where all the ORFs in a sequence are located, and most DNA sequences have many ORFs on both strands, although most of them are short. The region shown in Figure 2.44 contains many ORFs, but only the longest, in frame 6, is likely to encode a polypeptide. However, the presence of even a long ORF in a DNA sequence does not necessari ly indicate that the sequence encodes a protein, and fairly long ORFs often occur by chance. Furthermore, it has become evident recently that even very short ORFs can encode short peptides with important biological functions.

If an ORF does encode a polypeptide, it will begin with a TIR, but as discussed above, TIRs are sometimes difficult to identify. Clues to whether an ORF is likely to encode a protein may come from the choice of the third base in the codon for each amino acid in the ORF. Because of the redundancy of the code, an organism has many choices of codons for each amino acid, but each organism prefers to use some codons over others (see “Codon Usage” above) (Table 2.2).

A more direct way to determine if an ORF actually encodes a protein is to ask which polypeptides are made from the DNA in an in vitro transcription-translation system. These systems use extracts of cells, typically of E. coli, from which the DNA has been removed but the RNA polymerase, ribosomes, and other components of the translation apparatus remain. When DNA with the ORFs under investigation is added to these extracts, polypeptides can be synthesized from the added DNA. If the size of one of these polypeptides corresponds to the size of an ORF on the DNA, the ORF probably encodes a protein. Another way to determine if an ORF encodes a protein is to make a translation fusion of a reporter gene to the ORF and to determine whether the reporter gene is expressed (see below).