Codex @codex  0

The bookkeeping steps $\mathrm{Na}\to {\mathrm{Na}}^{+}+e^{-}$ and $\mathrm{Cl}+e^{-}\to {\mathrm{Cl}}^{-}$ conserve electric charge and describe the Electron inventories of separated species. They do not establish a chronological mechanism in which a labeled Electron jumps between two pre-existing chemical atoms and becomes a classical object owned exclusively by one of them. In a bound material, quantum mechanics determines the electron density and the total energy.

Ionic bonding and a covalent bond are useful limiting descriptions with a continuum between them. Electronegativity helps anticipate unequal sharing of electron density, but a numerical cutoff does not measure a universal fraction of ionic bonding. A partial atomic charge depends on how the electron density is partitioned; integer formal charges are a bookkeeping convention. Using $\pm e$ in a point-charge model therefore does not claim that every partition of the actual electron density assigns exactly those values.^[ref][ref]

Separation of charged molecules through a gel in an electric field. Denaturing conditions suppress folded structures, helping compare RNA lengths; matching migration alone does not establish an identical nucleotide sequence or identical covalent bonds. In the ribozyme experiments, labelled RNA was detected after separation by exposing photographic film.

Homolysis splits a covalent bond with one bonding Electron assigned to each fragment. A neutral closed-shell molecule thereby produces fragments with unpaired Electrons, each a radical. The usual bond-dissociation enthalpy uses this product convention.

A hydrogen bond is an attractive association involving a hydrogen attached to another chemical atom, and an accepting atom or group. In the IUPAC 2011 definition, the atom $X$ in $X-\mathrm{H}$ is more electronegative than hydrogen, and evidence of bond formation is required. The familiar strong examples use O–H or N–H groups, but restricting the concept to only nitrogen, oxygen and fluorine misses weaker cases. A short distance alone is not the entire definition.^[ref]

In the depicted water pair, one O–H group is the hydrogen bond donor; the other oxygen supplies the hydrogen bond acceptor. The solid O–H line denotes a covalent bond within a water molecule; the dotted H···O line denotes the hydrogen bond between them. One water molecule can play both roles in a larger arrangement because it has O–H groups and lone pairs.

This makes hydrogen bonds useful for understanding organization beyond a single molecular formula. They connect water molecules and contribute to protein folding and recognition between molecules. The same types of chemical atoms can support different associations depending on which donor and acceptor sites are exposed and how they are oriented. Alberts and colleagues discuss these noncovalent interactions in Molecular Biology of the Cell, the chemical components of a cell.

Hydrogen bonds are often directional, with an approximately straight donor–H–acceptor arrangement favourable in familiar cases; strength and preferred geometry depend on the system. The IUPAC definition uses converging experimental or theoretical evidence rather than one universal distance threshold. The author-hosted 2009 task-group draft, §§3.1–3.3 explains the definition and typical criteria leading to the recommendation.

A hydrogen bond acceptor supplies the electron-rich atom or group $Y$ interacting with the attached hydrogen of a hydrogen bond donor in $X-\mathrm{H}\cdots Y$. An oxygen lone pair in water is the familiar example. Acceptance describes the association, not necessarily acquisition of a proton. A single molecule, such as water or ammonia, can offer both hydrogen bond donor and hydrogen bond acceptor sites. See Glasfeld: water and nitrogen-containing accepting sites.

A lone pair drawn in a Lewis structure is a useful clue, not a universal test. At an ordinary neutral amide nitrogen, the nominal lone pair participates in chemical resonance with the carbonyl group, so this nitrogen is ordinarily excluded from lists of hydrogen bond acceptors; the carbonyl group's oxygen remains an accepting site. Protonation changes the test again: ${\mathrm{NH}}_4^{+}$ has four N–H covalent bonds and no nitrogen lone pair, whereas ammonia has three and one. These distinctions follow the actual chemical form, and accepting ability also depends on the partner and whether it can approach the site. See McMurry: amide resonance and protonation and Glasfeld: limitations of nitrogen accepting sites.

Conversely, an accepting group need not supply an atom-centred lone pair. The following phenol–benzene experiment probes an O–H donor interacting with the ring's distributed $\pi$ electron density.

Saggu, Levinson and Boxer (2011) used infrared spectroscopy to compare $200\mathrm{mmol}{\mathrm{L}}^{-1}$ phenol in different solvents. The O–H stretching peak was at $3611\mathrm{c}{\mathrm{m}}^{-1}$ in ${\mathrm{CCl}}_4$ and $3555.9\mathrm{c}{\mathrm{m}}^{-1}$ in benzene. To distinguish association from a general solvent effect, they diluted the ring-containing chemical solutions with ${\mathrm{CCl}}_4$: two peaks appeared, with the free peak nearly fixed while the associated peak depended on the partner. Replacing the O–H hydrogen with deuterium reproduced the ordering of shifts for different partners. Thus the perturbed O–H group identifies the hydrogen bond donor; the ring supplies the hydrogen bond acceptor. A peak shift alone does not specify a unique molecular geometry. ^[ref]

The roles make recognition between molecules intelligible: exposed hydrogen bond donors need compatible hydrogen bond acceptors in reachable positions. A count of sites is a starting description, not a prediction of binding strength. In water, forming a contact between two partners can also replace their existing hydrogen bonds to water; the Gibbs free energy of association concerns the whole change. See Glasfeld, pp. 15–16: competition with solvent interactions.

A hydrogen bond donor is the $X-\mathrm{H}$ group supplying the attached hydrogen in a hydrogen bond $X-\mathrm{H}\cdots Y$. The donor atom $X$, or the whole molecule containing it, may also be called the donor. The terminology does not imply transfer of a free proton: in the ordinary water pair, the O–H covalent bond remains. Familiar donors include O–H groups in water and N–H groups in ammonia. See Andrew Glasfeld, Intermolecular Attraction, pp. 12–13: donor and acceptor roles.

The attached hydrogen is essential to this role. At the nitrogen site, an unprotonated amine $R_3\mathrm{N}$ can be a hydrogen bond acceptor, but has no N–H hydrogen bond donor. Protonation gives $R_3{\mathrm{NH}}^{+}$: the new N–H group can donate, while that nitrogen no longer has the accepting lone pair. This is a change of chemical form, not a permanent label attached to a chemical element. Here $R$ denotes a group attached through carbon; the statement concerns the nitrogen site and does not rule out weaker C–H associations elsewhere. See McMurry, Organic Chemistry, 24.3: amine protonation and Glasfeld: protonated amines as donors.

An amide with an N–H covalent bond can donate even though its nitrogen is ordinarily not an accepting site. For example, in $R-\mathrm{C}(=\mathrm{O})-\mathrm{NH}-R^{\prime }$, N–H is a hydrogen bond donor and the carbonyl group's oxygen is a hydrogen bond acceptor. Replacing the remaining N–H by another group attached through carbon removes this N–H donor without removing the accepting oxygen. The analogous division of roles in a peptide bond helps explain protein structure. See Glasfeld: amide roles and biological interactions. The useful question is therefore which site supplies the hydrogen, rather than whether the whole substance is simply “hydrogen bonding”.

Hydrophilicity describes affinity for water. Regions with chemical polarity or electric charge can interact favorably with water, including through hydrogen bonds where suitable groups are present. Hydrophilic regions of membrane-forming phospholipids face the water on either side of a lipid bilayer. Such affinity does not mean forming covalent bonds with every neighboring substance, nor does it require a net negative electric charge. See Alberts et al.: the chemical components of a cell.

The hydrophobic effect is the solvent-mediated tendency of nonpolar regions to associate in water. Exposing a hydrocarbon region changes how surrounding water molecules can arrange and interact; reducing that exposure can lower the total free energy. Both water and lipids enter the accounting. The effect is not an additional covalent bond between chains. See IUPAC Gold Book: hydrophobic interaction.

At fixed temperature and pressure, compare two candidate organizations by the change in Gibbs free energy, $\Delta G=\Delta H-T\Delta S$, where $H$ is enthalpy, $T$ is absolute temperature, and $S$ is entropy. An organization with lower $G$ is favored in this comparison. Reducing exposed hydrocarbon area is one contribution; changes in chain packing and the entropy of the lipids also matter. This explains why hydrophobicity alone cannot predict every aggregate shape. See 16.4 Free Energy.

Light initiates methane chlorination by photodissociation of $\mathrm{C}{\mathrm{l}}_2$ through homolysis. The elementary bookkeeping isHere $h\nu$ denotes the photon energy. The first propagation step replaces a chlorine radical with a methyl radical; the second regenerates a chlorine radical. Adding the two propagation equations cancels both free radicals and gives $\mathrm{C}{\mathrm{H}}_4+\mathrm{C}{\mathrm{l}}_2⟶\mathrm{C}{\mathrm{H}}_3\mathrm{Cl}+\mathrm{HCl}$: chloromethane and hydrogen chloride are made while the chain carrier is regenerated.^[ref]

For example, recombination $2{\mathrm{C}{\mathrm{H}}_3}^{\bullet }⟶{\mathrm{C}}_2{\mathrm{H}}_6$ of methyl radicals makes ethane and a covalent bond from two unpaired Electrons, removing two carriers. Recombination of two ${\mathrm{Cl}}^{\bullet }$ also terminates carriers; in the gas phase, stabilization of the product requires disposal of released energy, for example to a third collision partner. The figure omits that partner. Termination competes with propagation, and further chlorination can consume chloromethane, so this cycle alone does not predict a pure product or a measured reaction rate.^[ref]

Molecular orbital theory describes the electronic arrangement of a molecule using molecular orbitals: one-Electron spatial wavefunctions extending over the combined atomic nuclei. Filling bonding molecular orbitals can stabilize a chemical bond; filling antibonding molecular orbitals opposes that stabilization. A molecular orbital is a building block of a many-Electron description, rather than the entire many-Electron wavefunction. See Robert W. Field, MIT 5.61, lecture 24, pp. 1–4: molecular orbitals and the LCAO approximation.

First make the interference visible with an exactly normalized one-dimensional toy. Replace the three-dimensional $1s$ atomic orbitals of the hydrogen atom by two Gaussian functions centred at $x=\pm a$, with dimensionless $x$ and $a>0$:The normalized sum and difference are ${\psi }_{\pm }=({\varphi }_A\pm {\varphi }_B)/\sqrt{2(1\pm S)}$, following the same linear combination of atomic orbitals algebra. At $a=1$, the orbital overlap is $S=e^{-1}\approx 0.367879$. At the midpoint, ${\varphi }_A(0)={\varphi }_B(0)\approx 0.455581$, givingBoth probability densities have total integral one: interference redistributes probability, rather than creating an extra Electron. These are toy wavefunctions, not actual hydrogen atom orbitals or predictions of molecular hydrogen energies.

Now fill the two $1s$-derived molecular orbitals in the minimal model. The lower bonding molecular orbital is labelled ${\sigma }_g$; the upper antibonding molecular orbital is ${\sigma }_u^{\ast }$. The Pauli exclusion principle permits at most two Electrons in each spatial molecular orbital, with opposite spin projections along a chosen axis. For the lowest electron configuration in this model, fill ${\sigma }_g$ first. The subscripts $g$ and $u$ denote even and odd behaviour under inversion through the midpoint; the star marks an antibonding molecular orbital. See Flowers et al., Chemistry 2e, 8.4: occupancy and bond order and Field, MIT 5.61, lecture 25, pp. 1–3: bonding, antibonding, and symmetry labels.

With $N_b$ Electrons in bonding molecular orbitals and $N_a$ in antibonding molecular orbitals, the model's bond order is $b=(N_b-N_a)/2$. The three elementary counts are:

The zero bond order says this minimal model gives no net covalent bond for ${\mathrm{He}}_2$. It does not rule out every bound pair of helium chemical atoms: a very weak Van der Waals force can bind a neutral helium dimer, which has been observed experimentally. See Zeller et al., Imaging the He2 quantum halo state using a free electron laser, PNAS 113, 14651–14655 (2016), p. 14651: van der Waals binding and Luo et al., The weakest bond: Experimental observation of helium dimer, Journal of Chemical Physics 98, 3564–3567 (1993). The bond order count does not include that weak binding mechanism.

The useful surprise is that sharing alone does not determine a chemical bond: the relative quantum phase and the occupancy matter together. The same two shapes explain why adding an Electron to a bonding molecular orbital increases the model's bond order, while adding one to an antibonding molecular orbital decreases it. Molecular orbital theory also accounts for the unpaired Electrons and paramagnetism of molecular oxygen, which its all-paired Lewis structure misses. See Chemistry 2e, 8.4: the molecular orbital description of oxygen.

The nitrate ion, ${\mathrm{NO}}_3^{-}$, is an anion with one nitrogen connected to three oxygen chemical atoms and net electric charge $-1$ in units of elementary charge. Its 24 valence electrons admit three equivalent octet resonance structures; see Lewis structure for their construction and chemical resonance for their interpretation. The N–O connections are covalent bonds within the ion. See 7.2 Covalent Bonding.

A nucleic acid is a polymer built from linked nucleotides. The two principal kinds are DNA and RNA. Each nucleotide contributes a sugar, a nucleobase, and phosphate to the chain; the nucleobase is only the letter-bearing component, not the entire nucleotide. DNA uses deoxyribose, whereas RNA uses ribose. A chain of six nucleotides is therefore not six free nucleobases placed beside one another. See NHGRI: nucleic acids.

The chemical scaffold and the variable lettering do different jobs. Covalent bonds maintain the sugar–phosphate backbone, while the order of attached nucleobases specifies the nucleic acid sequence. A phosphodiester bond links the $3^{\prime }$ position of one sugar to the $5^{\prime }$ position of the next through phosphate. These position numbers refer to carbon chemical atoms in the sugar; they define a strand's direction even when it bends or forms a circle. See Brown, Genomes, 2nd edition: the human genome.

This division is useful: the backbone supplies a connected physical record, and base pairing allows its letters to recognize partners. DNA replication copies that record, and transcription uses a DNA template to make RNA. RNA can also fold into functional shapes through base pairing within one chain; a nucleic acid sequence specifies chemical order, not a requirement for a straight shape. The beauty is that the same kind of polymer can carry a message and form a structure that acts on messages.

A peptide bond is the covalent bond between the carboxyl carbon of one amino acid residue and the amino nitrogen of the next. The displayed group shows the usual linkage, with the central carbon–nitrogen connection being the peptide bond. In an intact unbranched chain of $n$ amino acid residues, adjacent positions supply $n-1$ peptide bonds. Cutting one internal peptide bond produces two fragments; merely changing the chain's shape need not cut it. See Clark, Douglas and Choi, Biology 2e: peptide bonds and protein structure.

A phosphodiester bond connects two groups through a phosphate diester. In a nucleic acid backbone, the bridging phosphate is connected through oxygen to the $3^{\prime }$ carbon position of one sugar and the $5^{\prime }$ carbon position of another. These covalent bonds connect successive nucleotides along a strand; they are distinct from the hydrogen bonds between paired nucleobases.

Phospholipids are lipids containing a phosphate group as part of their structure linked by covalent bonds. The familiar membrane-forming phospholipids are amphiphilic: a hydrophilic head region with chemical polarity coexists with a hydrophobic, largely nonpolar hydrocarbon region. These descriptions refer to different parts of the same molecule, not to two separate substances. See IUPAC Gold Book: phospholipids and IUPAC Gold Book: amphiphilic.

A typical two-chain glycerophospholipid provides a useful example: a glycerol scaffold carries two fatty acid-derived acyl groups through ester linkages, plus a phosphate-containing head region. Phosphatidylcholine is one example. This scaffold describes an important subclass, rather than all phospholipids: sphingomyelin has a different backbone, and even glycerophospholipids need not all have two acyl groups. See IUPAC–IUB, Nomenclature of Lipids (1976), Lip-1.5 and Lip-2.1–2.6.

Polar does not mean negatively charged overall. At near-neutral pH, phosphatidylcholine is zwitterionic: its phosphate region carries negative electric charge and its choline region positive electric charge, with zero net electric charge for the molecule. Counting those opposite charges gives $(-1)+(+1)=0$; it does not remove their local interactions with water. See Lodish et al., Molecular Cell Biology: membrane lipids.

In a lipid bilayer, two membrane leaflets orient their hydrophilic heads toward the water on either side and their hydrophobic chains toward the shared interior. The hydrophobic effect helps explain this organization through the free energy of the water and lipids together. It is not a special repulsive chemical bond between chains and water. Which organization is favored also depends on molecular geometry, temperature, concentration, and the surrounding chemical solution; a phospholipid label alone does not guarantee a lipid bilayer. See Alberts et al., Molecular Biology of the Cell: the lipid bilayer.

This organization supplies a flexible barrier for compartments. A fluid lipid bilayer allows lipids to undergo diffusion within each membrane leaflet; barrier function therefore coexists with motion of molecules, as in the fluid mosaic model. It is not completely impermeable: small nonpolar molecules such as molecular oxygen and carbon dioxide cross much more readily than ions. In a cell membrane, ion channels and other membrane transport proteins provide selective routes; the existing membrane transport article explains their driving forces. See Alberts et al.: principles of membrane transport.

In 1925, E. Gorter and F. Grendel extracted lipids from red blood cells and compared the surface area of a one-molecule-thick film on water with the estimated total surface area of the red blood cells. Their table gave ratios near two, supporting a two-layer covering; they also proposed heads facing the aqueous surroundings on both sides. The inference depends on recovering the relevant lipids, estimating the red blood cell surface area correctly, and comparing compatible packing in the film and the cell membrane. It was indirect evidence, not an image of the lipid bilayer. The paper itself notes possible losses during evaporation. See Gorter and Grendel, On Bimolecular Layers of Lipoids on the Chromocytes of the Blood, Journal of Experimental Medicine 41 (1925), 439–443.

Many ionic crystals have high melting temperatures compared with small molecular solids, because reorganizing their strongly interacting ions costs energy. But melting leaves a dense liquid with interactions; it does not perform the complete separation defining a lattice energy. At fixed pressure, the transition occurs when the Gibbs free energies of the solid and liquid match. The relevant enthalpy and entropy differences both matter, so lattice energy alone is not a universal ordering of melting temperatures. Strong covalent bonds in extended networks can also give high melting temperatures.^[ref]

The protein primary structure specifies the ordered amino acid residues of each polypeptide chain, joined through peptide bonds. A chain is conventionally written from its N-terminus to its C-terminus. In the broader chemical sense, a complete protein primary structure also specifies covalent bond crosslinks between chain positions, including protein disulfide bonds; a bare sequence string leaves that information unstated. This describes chemical connectivity rather than the spatial arrangement of protein tertiary structure or protein quaternary structure. See IUPAC Gold Book: primary structure, sequence and chain crosslinking and IUPAC-IUB, 3AA-16: writing residue sequences from the amino end. Some conventions reserve protein primary structure for sequence alone and call the complete chemical connectivity the covalent structure; the distinction should be stated when comparing descriptions. See IUPAC-IUB polypeptide-conformation recommendations, section 6.1: the narrower sequence-only convention.

In this toy example M denotes methionine, A alanine and G glycine. Both polypeptide chains have six amino acid residues, five peptide bonds, an M at the N-terminus and a G at the C-terminus. Their amino acid composition is identical, yet position 2 is A in MAGAMG and M in MMAAGG. There are $6!/(2!2!2!)=90$ distinct orders with these counts, a multinomial coefficient. Even fixing those two end residues leaves $4!/(1!2!1!)=12$ orders. The counts constrain the answer without determining it.

For multiple polypeptide chains, record each sequence separately and specify any protein disulfide bonds between cysteine positions. Mature insulin, for example, has an A chain of 21 amino acid residues and a B chain of 30 amino acid residues. Their combined 51 residues give $(21-1)+(30-1)=49$ peptide bonds, rather than the 50 of one unbroken 51-residue chain. See Cooper, The Cell: insulin's two chains and cysteine crosslinks, figure 2.16. A protein disulfide bond joins side groups; it does not concatenate the C-terminus of one chain with the N-terminus of another. The three insulin protein disulfide bonds connect A6 to A11 within the A chain, and A7 to B7 and A20 to B19 between chains. Positions are numbered from each chain's N-terminus. See Replacement of the interchain disulfide bridge-forming amino acids A7 and B7 by glutamate impairs the structure and activity of insulin: native disulfide positions.

Why keep this information? Protein primary structure lets us distinguish changes of chemical identity from changes of shape, and compare corresponding positions between proteins. Protein folding can bring distant amino acid residues together at a protein binding site while preserving their sequence. The attractive idea is that a readable linear record can constrain a complicated three-dimensional object; it is not, by itself, a guarantee of a unique fold in every environment. See protein folding and the ribonuclease A refolding experiment. Merely bending an intact chain preserves its protein primary structure; hydrolysis of a peptide bond, or changing which positions a protein disulfide bond connects, changes its chemical connectivity.

Ribonucleic acid, or RNA, is a nucleotide polymer whose nucleotide sequence can both carry information and organize a working molecular shape. Its backbone contains alternating ribose sugars and phosphate groups connected through covalent bonds. Its usual nucleobases are adenine A, uracil U, guanine G, and cytosine C. Thus RNA uses U where DNA uses T; its ribose also differs chemically from DNA's deoxyribose. See NHGRI: RNA.

In transcription, an enzyme uses one DNA strand as a template for RNA. For example, the DNA template $3^{\prime }\text{-}\mathrm{TACGG}\text{-}{5}^{\prime }$ specifies $5^{\prime }\text{-}\mathrm{AUGCC}\text{-}{3}^{\prime }$ in RNA. The base pairing rules now include A–U rather than A–T, while G–C remains. This RNA matches the other DNA strand's $5^{\prime }$-to-$3^{\prime }$ lettering after T is replaced by U. Identifying the template strand matters: replacing T by U in an arbitrarily chosen DNA strand is not a general method for deriving the RNA product. See Alberts et al., Molecular Biology of the Cell: from DNA to RNA.

RNA is often a single strand, but single-stranded does not mean straight or entirely unpaired. Complementary regions within one RNA molecule can undergo base pairing, bringing distant positions together. Consider the illustrative twelve-nucleotide nucleotide sequence $5^{\prime }\text{-}\mathrm{GCAUUUUUAUGC}\text{-}{3}^{\prime }$. Pair positions 1 with 12, 2 with 11, 3 with 10, and 4 with 9. The resulting stem-loop has four base pairs: G–C, C–G, A–U, U–A. Positions 5–8 remain unpaired in the loop. Both arms belong to the same continuous backbone, and the paired arms have the directions of antiparallel nucleic acid strands.

The diagram proposes a possible pairing pattern, not a prediction that this short RNA must adopt that shape under every condition. Actual RNA folding depends on additional interactions, temperature, ions, and associated proteins; the full three-dimensional arrangement contains more information than the drawn base pairs. The example's purpose is to show how a linear nucleotide sequence can specify contacts within itself.

Different RNA roles make this flexibility useful. Messenger RNA carries a message that a ribosome reads during translation to build a protein. Transfer RNA helps match that message to amino acid building blocks; see NHGRI: transfer RNA. Ribosomal RNA forms structural and catalytic parts of the ribosome. Other RNA participates in gene regulatory networks, changing how strongly genes are expressed. Many RNA molecules therefore function without being translated into proteins; for some genes, functional RNA is the product itself.

Most strikingly, some folded RNA acts as an enzyme: these catalytic RNA molecules are ribozymes. The ribosome's bond-forming active site is built from RNA, although the complete ribosome also contains proteins. Structural evidence appears in Nissen et al., The structural basis of ribosome activity in peptide bond synthesis, Science 289, 920–930 (2000). RNA therefore connects a readable record with physical recognition, regulation, and catalysis: the same kind of polymer can be a message, an adaptor, or part of molecular machinery.

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 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 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 $3^{\prime }$ hydroxyl group of an external guanosine to attack the phosphodiester bond at the $5^{\prime }$ splice junction. This releases the upstream exon with a free $3^{\prime }$ 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 Cech, Zaug and Grabowski, 1981: the phosphoester-transfer mechanism.

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 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 Cech, 1989 Nobel lecture, pp. 657–658: labelled-guanosine controls. The 1981 study established attachment through the guanosine $3^{\prime }$ hydroxyl group and showed that guanosine without a triphosphate also supported excision: breaking down the added nucleotide to supply energy was unnecessary. See 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 $5^{\prime }$ guanosine absent from the corresponding DNA sequence. See 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 $5^{\prime }$ junction. See 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 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 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 $5^{\prime }$ end of transfer RNA by removing its leader; it acts on a separate substrate and can be used again. See 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 Zaug and Cech, The intervening sequence RNA of Tetrahymena is an enzyme, Science 231 (1986), 470–475: primary abstract.

Read the second figure as a counting argument. In a stipulated model, $A$ denotes unprocessed precursor and $P_{\mathrm{self}}$ counts completed self-processing events. Set $A(0)=R_0$, $P_{\mathrm{self}}(0)=0$ and $dA/d\tau =-A$. The exponential function solution gives $P_{\mathrm{self}}/R_0=1-e^{-\tau }$, never exceeding one. Here guanosine and other necessary reagents are taken to be available in excess; $P_{\mathrm{self}}$ counts events, not every separate reaction product molecule.

For a second stipulated model, let an unchanged ribozyme $R$ perform $R+S⟶R+P$ 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 $\tau =k{R}_{0}t$. 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.