Читать книгу Snyder and Champness Molecular Genetics of Bacteria - Tina M. Henkin - Страница 244

Probably the most convenient way to determine which of the possible ORFs on the two strands of DNA in a given region are translated into proteins is to make transcriptional and translational fusions to the ORFs. These methods make use of reporter genes, such as lacZ (β-galactosidase), gfp (green fluorescent protein), lux (luciferase), or other genes whose products are easy to detect. Figure 2.45 illustrates the concepts of transcriptional and translational fusions.

An ORF can be translated only if it is transcribed into RNA. Transcriptional fusions can be used to determine whether this has occurred. To make a transcriptional fusion, a reporter gene containing its TIR sequence but without its own promoter is fused immediately downstream of the promoter of the gene to be tested. If the promoter is active, and its gene is transcribed into mRNA, the reporter gene will also be transcribed, and the reporter gene product will be detectable in the cell. Transcriptional fusions also offer a convenient way of determining how much mRNA is made on a coding sequence. In general, the more reporter gene product that is made in the transcriptional fusion, the more mRNA was made that was directed by the upstream sequence. Translation of the mRNA depends on the activity of the TIR from the reporter gene and is usually consistent regardless of the identity of the upstream sequence. Examples of the use of transcriptional fusions in studying the regulation of operons are given in subsequent chapters.

Figure 2.45 Transcriptional and translational fusions to express a lacZ reporter (which encodes β-galactosidase). In both types of fusion, transcription begins at the +1 site at the porfA promoter upstream of the OrfA coding sequence, and the levels of mRNA generated are dependent on the activity of this promoter. (A) In a transcriptional fusion, both the upstream OrfA coding region and the downstream lacZ reporter gene are included in the mRNA, and translation can initiate from both TIRs. Only the TIR for lacZ is used to generate β-galactosidase protein. The translation of the upstream OrfA continues until it encounters a termination codon in frame, as indicated by the dotted line, and therefore the activity of the orfA TIR does not contribute to β-galactosidase synthesis. Levels of β-galactosidase indicate the activity of the poffA promoter. (B) In a translational fusion, the mRNA includes the orfA TIR but the lacZ portion lacks its own TIR. Translation of the mRNA initiates at the TIR upstream of OrfA to make a fusion protein containing the remaining portion of the upstream OrfA coding sequence fused (in frame) to the LacZ reporter protein. The prime symbols indicate that part of each protein may be deleted. Levels of β-galactosidase indicate the activity of both the poffA promoter and the orfA TIR.

In a translational fusion, the reporter gene lacks both a promoter and a TIR and its coding sequence is fused immediately downstream of the TIR of the gene under investigation; it is crucial that the two coding sequences are fused in such a way that they are translated in the same reading frame and there are no termination codons between them. Translation beginning at the TIR of the upstream coding sequence will proceed through the reporter gene coding sequence, resulting in a fusion protein that contains both polypeptide sequences. The reporter gene product can then be assayed as before to determine how much of the fusion protein has been made. The reporter gene product must retain its activity even when fused to the potential polypeptide encoded by the upstream ORF; otherwise, it will not be detectable. Many reporter genes have been chosen because their products remain active when fused to other polypeptides. Translational fusions are also often used to attach affinity tags to proteins to use in their purification.