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\b[RNA structure controls which ribosome-binding and decoding events are kinetically accessible.] A bacterial <messenger RNA> can base-pair with itself or other <RNA> molecules, forming alternative structures whose populations depend on sequence, ligand concentration, <temperature> and translating <ribosomes>. Its sequence consequently encodes both a polypeptide and part of the mechanism controlling production of that polypeptide.
At initiation, a stem can sequester the <Shine-Dalgarno sequence> or start region, reducing pairing with the anti-Shine-Dalgarno region of the small-subunit <ribosomal RNA> and preventing productive initiation. Opening that structure makes the same coding sequence more accessible. Start-site accessibility also matters in bacteria whose transcripts do not use a canonical <Shine-Dalgarno sequence>; not all bacterial initiation can be reduced to this one motif. The regulatory variable is often competition between folding, unfolding and ribosome binding rather than a permanently closed structure.
A <riboswitch> couples a ligand-binding <RNA> domain to an expression platform that can alternatively expose or hide a translation-initiation region. Binding of a metabolite can stabilize one structure without requiring a separate regulatory <protein>. Direct binding of thiamine derivatives to bacterial <messenger RNA> established an RNA-based route to metabolic regulation.<Thiamine derivatives bind messenger RNAs directly to regulate bacterial gene expression by Wade Winkler and others>[Abstract and pp. 952–956, direct metabolite binding by regulatory RNA]{ref} In an <RNA thermometer>, heating destabilizes a stem and exposes the initiation site. The FourU thermometer provides a concrete example in which uridines help pair with the ribosome-binding region of a heat-responsive transcript.<FourU: a novel type of RNA thermometer in Salmonella by Torsten Waldminghaus and others>[Abstract and results on the agsA 5′ untranslated-region thermometer]{ref} Sequence specificity and a biologically relevant transition range distinguish such a sensor from the nonspecific fact that all RNA eventually denatures on heating.
Trans-acting <small regulatory RNAs> can remodel target <messenger RNA> structure by base pairing. Some block initiation directly, often aided by the RNA-binding chaperone <Hfq>; others disrupt an inhibitory stem and activate translation. Pairing can also expose an <ribonuclease> cleavage site or recruit degradation machinery. Protein-binding regulators provide another route: a ribosomal protein can recognize a structured element in its own polycistronic transcript and repress excess ribosomal-protein production. Occupancy of a structured site thus links translation to stoichiometric demand.
In a <polycistronic messenger RNA>, translation of an upstream coding region can unwind a structure masking a downstream initiation site, producing <translational coupling>. During elongation, the <ribosome> can unwind many structures, but a sufficiently resistant downstream <RNA pseudoknot> or stem, combined with an appropriate slippery sequence, can promote <programmed ribosomal frameshifting>. This changes the reading frame and hence the ratio of distinct protein products. Structure can also influence recoding or pause positions; a predicted stable hairpin alone does not establish that a physiologically meaningful pause occurs.
RNA structure further couples translation to transcription. In the tryptophan <attenuation in bacterial gene regulation> mechanism, rapid or stalled translation of a leader peptide changes which alternative RNA stems form. This decides formation of a transcription terminator and therefore how much downstream transcript is available. The immediate switch is termination of <transcription>, even though ribosome movement and structured RNA generate the decision. Likewise, intrinsic terminator formation, transcript degradation and ribosome protection change protein output without necessarily changing translation efficiency per intact transcript.
A convincing experiment measures both transcript abundance and protein output. Fuse the regulatory region to a reporter while retaining a control promoter; compare ligand or temperature conditions with a construct lacking the proposed structural element. A mutation disrupting one side of a stem, followed by a compensatory mutation restoring pairing with a changed sequence, distinguishes pairing from a single nucleotide-specific contact. <RNA structure probing> can test accessibility directly, and <toeprinting> can report initiation-complex occupancy. Include controls for reporter folding, growth, RNA half-life and effects of the perturbation on the ribosome itself. These tests connect a structural transition to the appropriate mechanistic step rather than assuming every change in protein abundance is a change in initiation.
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