Difference between revisions of "Part:BBa I715052:Design"

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Trp Leader Peptide and anti-terminator/terminator

Design Notes

Anti-terminators occur in nature to regulate transcription of a variety of genes, such as bacterial operons and phage genes (Nudler et al., 2002). Transcriptional termination is caused by the formation of an RNA stem-loop structure, the terminator, that forces RNA polymerase to detach from the mRNA. An anti-terminator is another RNA stem-loop structure that precedes and inhibits the formation of the terminator and its stem-loop structure, thereby preventing termination (Nudler et al., 2002). Although the anti-terminator is also a stem-loop structure, it does not terminate transcription. If an anti-terminator forms, a terminator cannot form, and transcription continues. This transcriptional attenuation mechanism allows for regulation of transcription by either selectively terminating transcription prematurely or by allowing it to proceed. The choice between transcriptional termination or anti-termination will change what is transcribed and modify gene regulation in response to environmental conditions. Let’s look at a specific example to illustrate more clearly how anti-terminators work. The tryptophan operon in E. coli, trp, is regulated using an anti-terminator and encodes for genes that synthesize the amino acid tryptophan. The first important step in the transcription of the operon takes place at the leader peptide sequence, trpL. In bacteria, translation can begin while transcription is still under way. In this particular case, it is critical that transcription and translation are synchronized, such that a specific distance between the RNA polymerase and ribosome is set. RNA polymerase begins transcribing the mRNA until it produces a stem-loop structure which causes the polymerase to pause, denoted by the yellow region. The pause by RNA polymerase gives the ribosome time to start translating the trpL mRNA sequence into a short, non-functional leader peptide and to catch up with RNA polymerase. Once the ribosome has caught up to the RNA polymerase, the secondary structure that caused the RNA polymerase to pause is altered, and RNA polymerase continues transcription. This pausing of RNA polymerase causes transcription and translation to become coupled and to happen in synchrony (Landick et al., 1987). Before the leader peptide’s stop codon, and near the beginning of the anti-terminator sequence, are two sequential codons that code for tryptophan, shown in orange. If tryptophan concentrations are low, the ribosome will pause at these two tryptophan codons until tRNAs carrying tryptophan arrive. Meanwhile, RNA polymerase moves further downstream, elongating the mRNA molecule. The elongated mRNA forms an anti-terminator stem-loop structure which prevents the formation of a terminator stem-loop. The RNA polymerase continues mRNA elongation, the ribosome resumes once two tRNATrp arrive and the entire trp operon is transcribed and translated (Landick et al., 1987). Conversely, when tryptophan is abundant, the ribosome does not pause at the pair of tryptophan codons because tRNATrp are plentiful; translation continues unabated, and RNA polymerase does not distance itself from the ribosome. As the ribosome advances, it prevents the formation of the anti-terminator stem-loop structure through steric interactions. The lack of an anti-terminator structure leads to the formation of a new stem-loop structure, the transcriptional terminator, and causes the attenuation of the transcription. In this way, E. coli will not transcribe the trp operon unless it needs more tryptophan. The ribosome pauses temporarily only when there is a shortage of tryptophan, triggering the eventual synthesis of enzymes that will produce more tryptophan (Landick et al., 1987).

Source

PCR of genomic sequence

References