Ribosome Site Accessibility — What Is Base-Pairing Over Your RBS
Find every stretch of a transcript that can pair over the Shine-Dalgarno sequence or the start codon, with the length and melting temperature of each duplex — the mechanism behind every translational switch, and the first thing to check when a construct is silent.
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A construct that transcribes fine and makes no protein has a short list of causes, and the first one to rule out is the cheapest to check: something in the transcript is base-paired over the ribosome binding site. It is also the mechanism every engineered translational switch runs on — a riboswitch, a toehold switch, an RNA thermometer and an antisense repressor all work by pairing the Shine-Dalgarno sequence and the start codon into a stem in one state and freeing them in the other. So the same question starts both jobs: what in this transcript is complementary to the ribosome's footprint, and how strongly. This finds every such stretch, extends each duplex as far as the pairing holds, gives its nearest-neighbour melting temperature, and says which ones actually cover the SD core or the start codon rather than merely sitting nearby. It also reports the window 5' of the site where a sensor domain can be inserted without touching the site itself. What it does NOT do is fold anything: this is base-pairing arithmetic over the letters, exact and checkable, and whether a given stem wins in the ensemble at 37 °C is a different question with a different tool. It reports no switching ratio either, and the reason is written into the tool rather than left implied.
Every stretch of the transcript that is reverse-complementary to the ribosome's footprint, with the Tm of each duplex. This is the mechanism every translational switch runs on, and the first thing to check when a construct is silent for no obvious reason. Base-pairing arithmetic, not a fold.
How to use the Ribosome Site Accessibility tool
- 1Paste the transcript — the 5' UTR plus at least the start of the coding sequence.
- 2If there are several start codons, pin the one you mean; otherwise the first ATG, GTG or TTG anchors the footprint.
- 3Read the covered/uncovered verdict first, then the table: arm position, stem length, loop size and Tm.
- 4For a switch, use the insertion window — it is the region 5' of the SD where an aptamer or toehold goes.
Frequently asked questions
Is this a folding prediction?
No, and the distinction is the reason it can be trusted. It finds stretches that are reverse-complementary to the ribosome's footprint and measures each duplex with the nearest-neighbour model — both are arithmetic over the letters, exact and re-derivable. Whether a particular stem actually forms at 37 °C against everything else the molecule could do is an ensemble question, and that is what the RNA folding tool answers. Use this to find the candidates and that to weigh them.
What is the footprint, and why −20 to +13?
It is the region the 30S initiation complex covers on the mRNA, measured by toeprinting, stated relative to the A of the start codon. It is an empirical fact about the ribosome rather than something computable from your sequence, so it is a constant here and it is named in the output.
Why does it not give me a switching ratio?
Because an honest one needs constrained folding of the ligand-bound conformation against a validated aptamer structure set, and neither exists here. A dynamic range quoted without them would be a number with nothing behind it — which is worse than no number, because it looks like an answer. What you get instead is every pairing that could hold the site shut, with its length and Tm, which is the input to that judgement rather than a substitute for it.
My construct has no Shine-Dalgarno. Will it still work?
Yes, and that case is deliberately supported rather than refused — a missing or wrong SD is exactly when somebody needs this. The footprint is anchored on the start codon, the scan runs as normal, and a note says no SD core was found at a plausible spacing.
What counts as a stem worth worrying about?
The tool reports length and Tm rather than a verdict, because the threshold depends on your temperature and host. As orientation: a duplex under about 6 bp is unlikely to hold anything shut, and a GC-rich stem over 10 bp with a Tm above growth temperature is the shape that silences a construct. A stem covering the SD core matters more than one covering neighbouring bases, and the output flags which it is.
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