A device dubbed the “mother machine” enables real-time observation of mutagenesis in single bacterial cells.
Researchers have designed a screen to find unique molecules, called riboswitches, that determine whether transcription will proceed.
August 1, 2016|
© GEORGE RETSECK
Nature has evolved a staggering array of mechanisms for regulating gene expression, but few are so simple and elegant as the riboswitch. These RNA elements sit within the 5’ noncoding regions of bacterial messenger RNAs (mRNA) and regulate an mRNA’s own transcription or translation, depending on the switch’s conformation. In the case of a transcription-regulating riboswitch, for example, association of the switch with a particular ligand, such as a metabolite, can alter the switch’s structure and in turn terminate transcription.
Since the first riboswitches were identified in the early 2000s, “most of the known riboswitches have been discovered pretty much one by one by sequence comparisons,” says RNA expert Thomas Hermann of the University of California, San Diego. But while that approach works well for conserved riboswitches, it fails to identify those that are species-specific, Hermann says.
Term-seq, a new technique developed by molecular geneticist Rotem Sorek of the Weizmann Institute of Science in Rehovot, Israel, finds novel candidate riboswitches without the need for sequence comparisons. Adapter sequences are first ligated to the 3’ ends of bacterial RNAs and then used to initiate genome-wide deep sequencing. If a transcription-regulating riboswitch is present in a given RNA, then sequencing will reveal telltale, prematurely terminated versions of the transcript.
Using term-seq, Sorek and his team successfully identified 49 out of 53 (92 percent) of the known riboswitches present in Bacillus subtilis and identified a further 18 new candidate regulators. By treating B. subtilis and other bacteria with antibiotics and then performing term-seq, Sorek’s team also found a number of antibiotic-resistance genes under riboswitch control.
The method is limited to identifying riboswitches that regulate transcription rather than translation, says Hermann, but “since we’re going from no method to a method that works in [most] cases, it’s already pretty good.” (Science, 352:aad9822, 2016)
|RIBOSWITCH DISCOVERY METHOD||TECHNIQUE||HYPOTHESIS DRIVEN||EXPERIMENTAL VALIDATION REQUIRED?||RIBOSWITCH TYPES|
|Comparative analyses||Transcript sequences screened for similarity to existing riboswitches||Yes||Yes||Transcription and translation regulators|
|Term-seq||Presence of prematurely terminated transcripts identified during deep sequencing of 3’ adapter-ligated RNAs||
No. Unbiased search
of whole transcriptome
|Yes||Transcription regulators only|