= Ribozyme
{wiki}
A <ribozyme> is an <RNA> <molecule> whose folded structure provides <catalysis> of a <chemical reaction>. A <protein> <enzyme> builds its <active site> from an <amino acid> chain; a <ribozyme> builds a working <active site> from <RNA>, sometimes with metal <ions> and associated <proteins>. <Base pairing> can help recognize a <substrate>, but recognition alone does not establish <catalysis>: the <RNA> must promote chemical conversion. A passive <messenger RNA> template being read by a <ribosome> is not thereby a <ribozyme>. See https://herschlaglab.stanford.edu/s/39.pdf[Cech and Herschlag, Group I Ribozymes: Substrate Recognition, Catalytic Strategies, and Comparative Mechanistic Analysis, sections 1–3].
The surprise is that an information-bearing <polymer> can also perform chemistry. Examples include the self-splicing <group I intron> of Tetrahymena thermophila and the catalytic <RNA> component of bacterial <RNase P>, discussed below. The <ribosome> supplies an everyday example: its <peptide bond>-forming <active site> is made of <ribosomal RNA>, although the complete machine also contains <proteins>. Nissen and colleagues examined structures of a large <ribosome> subunit with <substrate> analogues and found <RNA> contacting those analogues at the catalytic centre. This supports an <RNA> <active site>, not a claim that isolated <ribosomal RNA> can carry out all of <biological translation>. See https://pubmed.ncbi.nlm.nih.gov/10937990/[Nissen, Hansen, Ban, Moore and Steitz, The structural basis of ribosome activity in peptide bond synthesis, Science 289 (2000), 920–930].
For the Tetrahymena <group I intron>, <RNA splicing> removes the <intron> and joins its flanking <exons>. The first chemical step uses the free $3'$ <hydroxyl group> of an external <guanosine> to attack the <phosphodiester bond> at the $5'$ splice junction. This releases the upstream <exon> with a free $3'$ <hydroxyl group> and attaches the <guanosine> to the <intron>. That newly freed <hydroxyl group> then attacks the <phosphodiester bond> at the other splice junction, joining the <exons> and releasing the linear <intron>. Each step exchanges one backbone connection for another; it is not simply separation of <base pairs>. See https://pubmed.ncbi.nlm.nih.gov/6101203/[Cech, Zaug and Grabowski, 1981: the phosphoester-transfer mechanism].
\Image[/ribozymes-splice-bond-map.png]
{title=<Group I intron> self-splicing: an external <guanosine> starts two exchanges of <phosphodiester bonds>}
{description=Original chemical-connectivity schematic, not an atomic structure or measured reaction trace. Orange junctions are the two bonds replaced; green junctions are the new bonds. The folded <RNA> <active site> and <magnesium> <ions> are omitted. The linear excised <intron> is shown before any subsequent circularization.}
The motivating experiment began with an unexpected control. As Cech recounts, Zaug purified a <ribosomal RNA> precursor from Tetrahymena using a detergent and phenol extraction, then compared test tubes with and without added <cell nucleus> extract. Incubation in salts and <nucleotides> released the same amount of small <intron> <RNA> in both cases, detected by <gel electrophoresis>. Repetition made a missing ingredient or accidental omission less plausible. Activity also survived treatments intended to destroy <proteins>. Nevertheless, absence of added extract did not by itself exclude a tightly attached, resistant <protein>, or prove that the small <RNA> had been newly excised rather than released from an already processed complex. See https://www.nobelprize.org/uploads/2018/06/cech-lecture.pdf[Thomas R. Cech, 1989 Nobel lecture, pp. 655–659: the unexpected control and alternative explanations].
An independent chemical readout addressed the latter problem. Cech mixed precursor <RNA> with <phosphorus>-32-labelled <guanosine> bearing three <phosphate> groups (GTP), and used <gel electrophoresis> followed by exposure to X-ray film to locate the label. Label appeared in the excised <intron> with <magnesium> present; it was absent when <magnesium> was omitted or labelled <ATP> replaced the labelled <guanosine> reagent. This experiment is described in https://www.nobelprize.org/uploads/2018/06/cech-lecture.pdf[Cech, 1989 Nobel lecture, pp. 657–658: labelled-guanosine controls]. The 1981 study established attachment through the <guanosine> $3'$ <hydroxyl group> and showed that <guanosine> without a triphosphate also supported excision: breaking down the added <nucleotide> to supply <energy> was unnecessary. See https://pubmed.ncbi.nlm.nih.gov/6101203/[Cech, Zaug and Grabowski, In vitro splicing of the ribosomal RNA precursor of Tetrahymena, Cell 27 (1981), 487–496: primary abstract]. Separately, sequencing and terminal analysis found an extra $5'$ <guanosine> absent from the corresponding <DNA> sequence. See https://pubmed.ncbi.nlm.nih.gov/7099968/[Zaug and Cech, The intervening sequence excised from the ribosomal RNA precursor of Tetrahymena contains a 5-terminal guanosine residue not encoded by the DNA, Nucleic Acids Research 10 (1982), 2823–2838]. Together, incorporation and sequence evidence support a new <covalent bond>, beyond a change in how <RNA> associates.
Kruger, Grabowski, Zaug, Sands, Gottschling and Cech's 1982 test removed dependence on a precursor purified from Tetrahymena. They cloned the <intron> and adjacent <exons> into a <DNA> template, made precursor <RNA> with purified bacterial <RNA polymerase> under conditions suppressing <RNA splicing>, and removed <proteins> by detergent–phenol extraction. Denaturing <gel electrophoresis> separated precursor species. Purified precursor was then incubated with the <guanosine> reagent, <magnesium> and salt; matched samples without this incubation supplied a precursor baseline. Their standard assay used $10\,\mathrm{mM}$ magnesium chloride, $0.1\,\mathrm{mM}$ of that <guanosine> reagent, $100\,\mathrm{mM}$ ammonium sulfate and a <buffer solution> at <pH> $7.5$, for $30$ minutes at <temperature> $37\,{}^{\circ}\mathrm C$, with specified variations in individual experiments. <Gel electrophoresis> and radioactive-film detection showed excised <intron> <RNA>; sequencing verified its $5'$ junction. See https://dosequis.colorado.edu/courses/methodslogic/papers/Cech1982.pdf[Kruger et al., Self-Splicing RNA, Cell 31 (1982), 147–157, figures 3–4 and Experimental Procedures].
Excision alone could leave disconnected <exons>. To test joining, the authors paired labelled <RNA> with <DNA> spanning the joined-<exon> sequence, removed exposed <RNA> with <enzymes>, and separated the protected pieces by denaturing <gel electrophoresis>. Spliced samples yielded protected <RNA> long enough to span the junction. See https://dosequis.colorado.edu/courses/methodslogic/papers/Cech1982.pdf[Kruger et al., 1982, figure 6: independent evidence for exon ligation]. The experimental logic matters: an <intron>-sized band identifies a candidate product, while sequence and junction assays test its chemical identity.
These controls support sufficiency of the prepared <RNA> under the assay conditions. They do not show that every <RNA> self-splices, or that <proteins> are dispensable inside a <biological cell>. Many <group I introns> use <proteins> to help attain an active fold. See https://herschlaglab.stanford.edu/s/39.pdf[Cech and Herschlag, Group I Ribozymes, section 6: protein facilitation]. Nor does a failed assay necessarily identify an inactive <nucleotide sequence>: unsuitable <ions>, <pH>, or <temperature> can prevent a working <active site> from forming.
The distinction between self-processing and repeated use became especially clear with bacterial <RNase P>. In 1983 Guerrier-Takada, Gardiner, Marsh, Pace and Altman separated the <RNA> and <protein> components of <RNase P> from two <bacteria>. Purified <RNA> cleaved precursor <transfer RNA> under conditions containing $60\,\mathrm{mM}$ <magnesium> <ions>, or $10\,\mathrm{mM}$ <magnesium> <ions> plus $1\,\mathrm{mM}$ spermidine. The <protein> components alone had no catalytic activity. At $5$–$10\,\mathrm{mM}$ <magnesium> without spermidine, both components were needed. Thus the comparison demonstrated an <RNA> <catalyst> while also explaining why <protein> assistance could matter under other conditions. <RNase P> produces the mature $5'$ end of <transfer RNA> by removing its leader; it acts on a separate <substrate> and can be used again. See https://pubmed.ncbi.nlm.nih.gov/6197186/[Guerrier-Takada et al., The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme, Cell 35 (1983), 849–857: primary abstract].
Self-splicing demonstrates <RNA> chemistry, but one precursor completing one rearrangement is not yet evidence for catalytic turnover: repeated conversion of separate <substrates> by a regenerated <catalyst>. The self-splicing precursor changes its own connectivity and incorporates external <guanosine>. In 1986 Zaug and Cech showed that a shortened Tetrahymena <intron> <RNA> could cleave and rejoin separate short <RNA> <substrates> in a sequence-dependent reaction. This demonstrated an <RNA> <enzyme> acting beyond its own self-processing. See https://pubmed.ncbi.nlm.nih.gov/3941911/[Zaug and Cech, The intervening sequence RNA of Tetrahymena is an enzyme, Science 231 (1986), 470–475: primary abstract].
\Image[/ribozymes-turnover-model.png]
{title=Stipulated <chemical kinetics> models: one self-processing event versus repeated <ribozyme> turnover}
{description=Original analytical illustration, with dimensionless time and amounts normalized by the initial <RNA> amount. Curves are calculated from the equations below, not experimental data or fits to Tetrahymena or <RNase P>. Equal exponential timescales are a chosen teaching convention; the figure does not compare their biological speeds.}
Read the second figure as a counting argument. In a stipulated model, $A$ denotes unprocessed precursor and $P_{\rm self}$ counts completed self-processing events. Set $A(0)=R_0$, $P_{\rm self}(0)=0$ and $dA/d\tau=-A$. The <exponential function> solution gives $P_{\rm self}/R_0=1-e^{-\tau}$, never exceeding one. Here <guanosine> and other necessary reagents are taken to be available in excess; $P_{\rm self}$ counts events, not every separate <reaction product> <molecule>.
For a second stipulated model, let an unchanged <ribozyme> $R$ perform $R+S\longrightarrow R+P$ on a separate <substrate> $S$. Assume the effective <law of mass action> $dS/dt=-kRS$, no reverse reaction, no <ribozyme> loss and no inhibited or long-lived bound states. Set $R=R_0$, $S(0)=5R_0$, $P(0)=0$ and $\tau=kR_0t$. Then $S/R_0=5e^{-\tau}$ and $P/R_0=5(1-e^{-\tau})$, with $S+P=5R_0$. At $\tau=1$, self-processing gives about $0.632$ completed events per initial <RNA>, whereas the regenerated <ribozyme> gives about $3.161$ <reaction product> <molecules> per initial <ribozyme>. More than one <substrate> conversion per initial <ribozyme>, with appropriate background and identity controls, is evidence for turnover. Product accumulation below that threshold alone cannot distinguish a reusable <catalyst> from one-shot chemistry. These models illustrate that distinction; actual <enzyme kinetics> can include folding delays, binding, saturation and product release.
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