REVEALING RIBOSWITCHES: RNA is isolated from bacteria and tagged with a 3’ adapter, to which a complimentary oligonucleotide binds for reverse transcription and sequencing. Sequence analysis then reveals those transcripts that have been fully transcribed versus those prematurely truncated. The reproducible presence of similarly truncated transcripts for a given gene suggests the presence of a riboswitch in the mRNA.© 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...
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 |
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