Genetics and Molecular Biology · Unit 4: From gene to protein, and its control

Transcription: From DNA to RNA

Transcription copies one strand of a gene into RNA. This lesson follows the reaction in E. coli, where one core polymerase and a sigma factor find promoters, and in the eukaryotic nucleus, where three polymerases share the work and Pol II needs a set of general transcription factors. You will be able to say how each system starts and stops, and to read a DNA sequence and name the template strand, the coding strand, the direction of travel and the RNA made.

Watch first Which Strand Does the Polymerase Read · 3:05

The death cap mushroom makes a toxin, alpha-amanitin, that blocks one enzyme in your cells at very low doses: RNA polymerase II, which copies genes into messenger RNA. Two other RNA polymerases in the same nucleus work on at the same dose.

Why would a nucleus need three enzymes to do one job? And why can a bacterium such as E. coli manage with one? Both questions are about transcription, the copying of a stretch of DNA into RNA. To answer them you need the reaction itself, then the two systems that run it, then a way to read a sequence and say which strand was copied.

The reaction: RNA copies one strand

RNA polymerase opens a short stretch of the double helix and uses one strand as a guide. That strand is the template strand (also called the antisense or noncoding strand). The polymerase lines up ribonucleoside triphosphates (ATP, GTP, CTP, UTP) by base pairing with the template, A opposite T, U opposite A, G opposite C, and joins each to the 3’ hydroxyl of the chain before it, releasing pyrophosphate. Three rules follow.

  • The RNA grows 5’ to 3’, as DNA does in DNA replication.
  • Because the RNA is antiparallel to the strand it pairs with, the polymerase reads the template 3’ to 5’.
  • The RNA has the same sequence as the other strand, the coding strand (nontemplate strand), with U in place of T.

Unlike DNA polymerase, RNA polymerase needs no primer. It can join the first two nucleotides itself. Think of the coding strand as the printed page and the template as its photographic negative: the polymerase reads the negative and prints a copy of the page.

Where to start is set by a promoter, a DNA sequence where the polymerase and its helpers bind. The first nucleotide copied into RNA is called +1. Bases toward the 5’ end of the coding strand are upstream and get negative numbers; bases past +1 are downstream. Where to stop is set by a terminator. Each gene uses only one strand as template, but neighboring genes can use opposite strands.

Bacteria: one core enzyme and a sigma factor

In E. coli one kind of core enzyme makes nearly all cellular RNA. It has five subunits: two alpha, one beta, one beta’ (beta prime) and one omega. Core can copy DNA, but it does not find promoters well. A sigma factor binds the core to form the holoenzyme, and sigma reads the promoter.

The housekeeping sigma of E. coli, sigma 70, recognizes two short elements on the coding strand. Their consensus, the most common base at each position across many promoters, is:

-35 element: 5’-TTGACA-3’ … about 17 bp … -10 element: 5’-TATAAT-3’

The -10 element is also called the Pribnow box, after David Pribnow, who in 1975 reported the sequence of a polymerase binding site at an early promoter of phage T7. Real promoters differ from consensus at several positions. A closer match usually means more initiation, but not always, since other DNA and other proteins also matter.

Initiation goes in steps. The holoenzyme binds double-stranded DNA (the closed complex), then melts about a turn of the helix around the -10 element and the start site to form the open complex, with the template strand placed in the active site. The enzyme then makes and releases many short RNAs while still sitting at the promoter, a stage called abortive initiation. Eventually one RNA grows long enough, and the polymerase leaves the promoter (promoter escape) and elongates. Textbooks often say sigma is released at escape. In single-molecule work with purified E. coli enzyme, a substantial share of elongating complexes kept sigma 70, so release is better called common than obligatory.

Other sigmas send the same core to other genes. In E. coli, sigma 32 directs it to heat-shock genes, so a temperature rise changes which promoters are used.

Because bacteria have no nucleus, ribosomes can start translating an mRNA while its 3’ end is still being made. Many bacterial mRNAs are polycistronic, carrying the coding sequences of several genes from one promoter (an operon, the subject of the lac operon), but not all are.

Eukaryotes: three polymerases and the general factors

The nucleus divides the work among three polymerases. In mammalian cells:

  • Pol I makes the large rRNA precursor (45S or 47S), later cut into 28S, 18S and 5.8S rRNA.
  • Pol II makes the precursors of mRNAs, most small nuclear RNAs (snRNAs) and the precursors of microRNAs.
  • Pol III makes tRNAs, 5S rRNA, U6 snRNA and other small RNAs.

Their sensitivity to alpha-amanitin differs, which is why the toxin in the opening blocks only some RNAs.

Pol II cannot find a promoter by itself. It needs the general transcription factors, named TFII plus a letter, which assemble with it at the core promoter into a preinitiation complex:

  • TFIID contains the TATA-binding protein (TBP) and other subunits, and binds the core promoter first.
  • TFIIA and TFIIB steady TBP on the DNA; TFIIB also helps set the start site and bridges to Pol II.
  • TFIIF arrives with Pol II; TFIIE then recruits TFIIH.
  • TFIIH has a translocase subunit (XPB) that helps open the DNA, and a kinase subunit (CDK7).

A TATA box (consensus TATAAA) sits about 25 to 30 bp upstream of the start site in promoters that have one. Most human promoters do not. At those, TFIID is placed through other core elements, such as the initiator that covers the start site.

The largest Pol II subunit ends in a tail called the CTD (carboxy-terminal domain), made of repeats of seven amino acids, YSPTSPS. Human Pol II has 52 repeats and budding yeast 26. As the polymerase starts, CDK7 phosphorylates serine 5 of the repeats, and during elongation serine 2 is phosphorylated by other kinases. The phosphorylated tail acts as a landing pad: it recruits the enzymes that cap the RNA and later those that splice it and cut its 3’ end (see RNA processing). Proteins that switch genes on, such as the ones at the ends of signaling pathways to the nucleus and the nuclear receptors, act largely by helping or hindering this assembly. They bind DNA with the kind of specific, regulated contacts described in protein binding and allostery.

Once it escapes, Pol II can travel fast. In one study of long genes in cultured human cells, the average rate was about 3.8 kb per minute. If a gene ran 114 kb at that constant rate, ignoring pauses:

time = length / rate = 114 kb / 3.8 kb per min = 30 min

That is elongation alone, not the time to a finished mRNA. Rates differ among genes and cell types.

Stopping: termination in each system

E. coli uses two kinds of terminator, and they overlap.

An intrinsic terminator is written into the RNA itself: a GC-rich stretch that folds back into a hairpin, followed by a run of U residues. The hairpin forms in the RNA exit channel while the U tract holds the RNA to the template by weak A-U pairs, and together they release the RNA. A Rho-dependent terminator needs Rho, an ATP-driven protein that binds the nascent RNA at a Rho utilization (rut) site, moves along the RNA toward the polymerase, and pulls it off. The two classes are less separate than the textbook split suggests: in an atlas of Bacillus subtilis terminators, Rho stimulated about 10% of the intrinsic ones.

In eukaryotes each polymerase stops its own way. For Pol II, termination is coupled to processing. After the polymerase copies the poly(A) signal, the enzyme CPSF73 cuts the RNA, and the RNA still attached to the polymerase is chewed 5’ to 3’ by XRN2. In the torpedo model XRN2 catches the polymerase and knocks it off. In the allosteric model the polymerase changes after the signal and slows. In cultured human cells, experiments that rapidly removed these factors support a combination: slowing beyond the poly(A) site lets XRN2 catch up. Pol III stops at a run of T residues in the nontemplate strand, though in budding yeast the run is not always sufficient and RNA structure and the helicase Sen1 assist.

Strands and directions: the sequence problem

top:     5'-TACGGATCCATGAAACGT-3'
bottom:  3'-ATGCCTAGGTACTTTGCA-5'
RNA:     5'-ACGUUUCAUGG-3'   (made from this region)

Step 1. Check the duplex. Pair each base: T with A, A with T, C with G and so on across all 18 positions. Every pair matches, so the two strands are complementary.

Step 2. Write each strand 5’ to 3’. The top already is: TACGGATCCATGAAACGT. Reverse the bottom: 5’-ACGTTTCATGGATCCGTA-3’.

Step 3. Find the strand that matches the RNA, with T for U. The RNA, ACGUUUCAUGG, matches the first 11 bases of the bottom strand read 5’ to 3’ (ACGTTTCATGG). It does not occur in the top strand. So the bottom strand is the coding strand.

Step 4. The template is the other strand. The top strand is the template. Check: the RNA pairs with the top strand antiparallel. RNA 5’-A pairs with the top strand’s 3’-T, RNA C with G, G with C, and so on leftward.

Step 5. Direction of travel. The polymerase reads the template 3’ to 5’. On the top strand, 3’ is at the right, so the polymerase moves right to left along the page. The RNA grows 5’ to 3’ in that same leftward direction, and its 5’ end lies toward the right of the duplex as written.

Two checks catch most mistakes. The RNA must be antiparallel to its template. And without the RNA or a promoter, a bare duplex does not tell you which strand is the template.

Template strand reader: flip the promoter⚙ original · interactive
Promoter
11 of 18

Rule to keep: a bare duplex does not tell you the template; the promoter, or the RNA, does. The polymerase reads the template 3′ to 5′, builds RNA 5′ to 3′, and the RNA matches the coding strand with U for T. The picture shows only the stretch drawn and is a sketch of the logic; the lesson does not draw a promoter, so its placement beside +1 here is not to scale.

Design problem

A newly found mammalian gene makes an RNA of unknown type. Design an experiment to decide which nuclear RNA polymerase transcribes it.

One way to do it

Use the different sensitivities of the three polymerases to alpha-amanitin. Isolate nuclei from the cells and run a nuclear run-on: give the nuclei labeled nucleotides so that polymerases already on genes extend their RNAs. Set up a series of alpha-amanitin concentrations spanning several orders of magnitude, from none through roughly 1 µg/mL to a few hundred µg/mL. (These values are orders of magnitude for mammalian cells and should be checked against your cell type.) Measure labeled RNA from the new gene and from three controls: an rRNA gene (Pol I), a protein-coding gene such as actin (Pol II), and a tRNA or 5S rRNA gene (Pol III).

In mammalian cells you expect the Pol II control to drop at low doses, around 1 µg/mL or less; the Pol III control to drop only at roughly 100 to 1000 times more; and the Pol I control to resist across the range. Match the new gene’s curve to one control. The same titration can be done by in vitro transcription in a nuclear extract with the gene as template.

Then confirm. Map the RNA’s 5’ end by primer extension or from RNA-seq data to locate the start site, and inspect the region upstream for promoter elements of the matching class. Finally, fuse that promoter region to a reporter gene and test whether its expression shows the same alpha-amanitin sensitivity.

How we measure it

In vitro transcription with purified components

You mix a DNA template, purified polymerase, chosen factors and labeled nucleotides, then measure the RNA made. Adding or removing one component tells you what it is needed for, as when sigma restored transcription of phage DNA or general factors let Pol II start at a promoter.

Column chromatography of enzyme activity

A cell extract is passed through a resin that holds proteins by charge, and fractions are collected and assayed. Separate peaks of activity show separate enzymes or separable parts, which is how sigma was split from core and how Pol I, II and III were first resolved.

Nuclear run-on assay

Nuclei are isolated and given labeled nucleotides, so polymerases already engaged on genes extend their RNAs a short distance. Measuring labeled RNA from a given gene reports how much it was being transcribed, and adding inhibitors such as alpha-amanitin shows which polymerase does it.

X-ray crystallography and cryo-EM

Crystals or frozen particles of a polymerase complex are imaged to build an atomic model. The models show where the template, the RNA and each factor sit, which turns a list of steps into a physical mechanism.

Finish line

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Practice and MCAT-style, with explained answers.

Next · Lesson 17

RNA Processing: Caps, Tails and Splicing

Why is a message copied from more than 5,000 nucleotides of DNA only 737 nucleotides long?

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Yeast RNA polymerase II — the enzyme that makes mRNA
Yeast RNA polymerase II — the enzyme that makes mRNA ● LIVE · RCSB PDB 1I50
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Loading live structure 1I50 from RCSB PDB…

Find the deep cleft between the two largest subunits, where the DNA lies, and the clamp that swings over it. The active site, with its magnesium ion, sits at the floor of the cleft.

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TATA-binding protein on a TATA box — how TFIID grips a promoter
TATA-binding protein on a TATA box — how TFIID grips a promoter ● LIVE · RCSB PDB 1YTB
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Loading live structure 1YTB from RCSB PDB…

TBP is shaped like a saddle that sits on the DNA. Look at how sharply the DNA bends under it; the bend helps assemble the other factors at the start site.

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E. coli RNA polymerase with sigma 70 — the bacterial holoenzyme
E. coli RNA polymerase with sigma 70 — the bacterial holoenzyme ● LIVE · RCSB PDB 4YG2
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Loading live structure 4YG2 from RCSB PDB…

Trace sigma across the outside of the core enzyme: its domains sit where they can reach the -10 and -35 elements of a promoter, about two turns of DNA apart.

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Human RNA polymerase III about to terminate — a run of Ts as a stop signal
Human RNA polymerase III about to terminate — a run of Ts as a stop signal ● LIVE · RCSB PDB 7FJJ
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Loading live structure 7FJJ from RCSB PDB…

Look for the nontemplate DNA strand with its run of T residues held in a narrow tunnel of the RPC2 subunit. The contact helps explain why Pol III stops at T runs.

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Richard Burgess

Richard Burgess

With Andrew Travers, John Dunn and Ekkehard Bautz, separated E. coli RNA polymerase into a core enzyme and a sigma factor, and showed that sigma restores transcription of phage DNA.

In the late 1960s Richard Burgess was a graduate student at Harvard University, purifying RNA polymerase from E. coli. When the enzyme was passed over a column of phosphocellulose, a resin that separates proteins by their charge, one protein came away from the rest. The enzyme that remained could still make RNA, but it copied DNA from the phage T4 poorly. Adding the separated protein back restored transcription of the phage DNA. In 1969 Burgess, Andrew Travers, John Dunn and Ekkehard Bautz reported the result in Nature. They called the stimulating protein sigma factor and the enzyme left behind the core. Later work supported the reading that sigma lets the polymerase begin at the right places, at promoters, while the core alone makes RNA but chooses where to start poorly. Splitting one enzyme into a part that copies and a part that chooses made it possible to ask whether a cell could swap the choosing part, and alternative sigma factors that direct the core to different sets of genes were later found in many bacteria.

✓ verbatim Burgess, Travers, Dunn & Bautz, Nature 221:43 (1969), 'Factor stimulating transcription by RNA polymerase'.
Robert Roeder

Robert Roeder

With William Rutter, separated eukaryotic DNA-dependent RNA polymerase activity into three forms, I, II and III; later his laboratory showed that purified Pol II needs added factors to start at a promoter.

Robert Roeder trained in William Rutter's laboratory at the University of Washington, where he earned a PhD in biochemistry in 1969. That year Roeder and Rutter reported in Nature that extracts of eukaryotic cells contain more than one DNA-dependent RNA polymerase. When the activity was run through an ion-exchange column, it came off in separate peaks, designated I, II and III. A bacterium has one kind of core polymerase; the nucleus, it turned out, has three. In the decade that followed, work from several laboratories, including Roeder's and Rutter's, tied each form to a class of RNA and showed that they differ in sensitivity to the toxin alpha-amanitin. In 1979 Roeder's laboratory reported that purified Pol II could not start correctly at a viral promoter by itself; it needed fractions from a cell extract. Those fractions were later separated into the general transcription factors, TFIIA, TFIIB and the rest. That made it possible to rebuild accurate Pol II initiation from defined parts.

✓ verbatim Roeder & Rutter, Nature 224:234 (1969), 'Multiple forms of DNA-dependent RNA polymerase in eukaryotic organisms'; Weil, Luse, Segall & Roeder, Cell 18:469 (1979).
Roger Kornberg

Roger Kornberg

His laboratory determined the atomic structure of yeast RNA polymerase II, alone and holding DNA and RNA; he received the 2006 Nobel Prize in Chemistry for studies of the molecular basis of eukaryotic transcription.

Roger Kornberg's laboratory at Stanford University set out to see RNA polymerase II atom by atom. The enzyme is large, with a dozen subunits in yeast, and making crystals of it that diffract well was the main obstacle. In 2001 Patrick Cramer, David Bushnell and Kornberg reported in Science a structure of a ten-subunit form of budding yeast Pol II at 2.8 Å resolution. A companion paper from the same laboratory, by Averell Gnatt and colleagues, showed the enzyme holding template DNA and a growing RNA chain. The structures showed a deep cleft that holds the DNA, a mobile clamp over it, a short stretch where the new RNA stays paired with the template, and a separate path by which the RNA leaves. In 2006 Kornberg received the Nobel Prize in Chemistry for his studies of the molecular basis of eukaryotic transcription. His father, Arthur Kornberg, had shared the 1959 Nobel Prize in Physiology or Medicine for work on the synthesis of DNA. The Pol II structures gave later work a frame in which to place each general factor and each step of the reaction.

✓ verbatim Cramer, Bushnell & Kornberg, Science 292:1863 (2001); Gnatt et al., Science 292:1876 (2001); Nobel Prize in Chemistry 2006 facts page, nobelprize.org.
Test yourself
Question 1 of 8 Score 0 / 0

Which component lets E. coli RNA polymerase begin transcription at promoters?
Sources & Integrity

Every structure, slide, simulation, and clip on this page is listed below with the source it came from. None of the 3D structures is drawn by hand or by AI: each one loads by accession ID from RCSB PDB or PubChem. This panel is generated from the lesson's own data file, so what it lists is what the page loads.

Molecular structures live data

  • Yeast RNA polymerase II — the enzyme that makes mRNA — RCSB PDB 1I50 · coordinates CC0 · Ten-subunit Saccharomyces cerevisiae Pol II at 2.8 Å, X-ray crystal structure (Cramer, Bushnell & Kornberg, Science, 2001)
  • TATA-binding protein on a TATA box — how TFIID grips a promoter — RCSB PDB 1YTB · coordinates CC0 · Yeast TBP bound to TATA-box DNA, X-ray crystal structure (Kim, Geiger, Hahn & Sigler, Nature, 1993)
  • E. coli RNA polymerase with sigma 70 — the bacterial holoenzyme — RCSB PDB 4YG2 · coordinates CC0 · E. coli sigma 70 holoenzyme, X-ray crystal structure (Murakami, J. Biol. Chem., 2013)
  • Human RNA polymerase III about to terminate — a run of Ts as a stop signal — RCSB PDB 7FJJ · coordinates CC0 · Human Pol III pretermination complex on a scaffold with seven dT, cryo-EM at 3.6 Å (Hou et al., Nature Communications, 2021)

Simulations

  • Template strand reader: flip the promoter · Original interactive — Biochemistrypedia (Deppmann Lab).

Scientist profiles & quotes

  • Richard Burgess — Burgess, Travers, Dunn & Bautz, Nature 221:43 (1969), 'Factor stimulating transcription by RNA polymerase'.
  • Robert Roeder — Roeder & Rutter, Nature 224:234 (1969), 'Multiple forms of DNA-dependent RNA polymerase in eukaryotic organisms'; Weil, Luse, Segall & Roeder, Cell 18:469 (1979).
  • Roger Kornberg — Cramer, Bushnell & Kornberg, Science 292:1863 (2001); Gnatt et al., Science 292:1876 (2001); Nobel Prize in Chemistry 2006 facts page, nobelprize.org.

Quotes are verbatim from the cited source. Profile text is original to this site and was checked against the sources cited.

Animated explainer

Which Strand Does the Polymerase Read · narration: an AI narrator voice (Gemini, Charon) reading narration written from this lesson's text · animation: AI-generated in code (Manim) by Claude, a schematic with no drawn molecular structures, plots computed from their equations · captions from the narration script.

Images & figures

This lesson carries no raster figures. Every structure is rendered live from RCSB PDB or PubChem, and every plot is drawn from its equation in the browser.

integrity Machines check the mechanics. The build confirms that every local file exists, that every question's answer key points at a real choice, and that any AI-generated scientific figure names the person who checked it; a weekly job re-resolves every accession ID against its database. Questions are original; MCAT-style items are written for this site and are not real AAMC questions. Banned sources (the Tymoczko textbook, publisher decks, copyrighted figures) are not used.

Like I'm five

Your cells keep their instructions in DNA, which stays safely put away. To use one instruction, a machine called RNA polymerase makes a copy of it in a slightly different material, RNA. The copy can go to where the work is done. Signs on the DNA tell the machine where to start copying and where to stop.
High school

Transcription is making an RNA copy of a gene. The DNA double helix opens, and RNA polymerase uses one strand, the template, to line up matching RNA nucleotides, with U in place of T. The new RNA has the same sequence as the other DNA strand, the coding strand. Polymerase starts at a stretch of DNA called a promoter and stops at a terminator. In bacteria one kind of polymerase does all of this, helped by a protein called sigma that finds promoters. In the nucleus of your cells there are three polymerases, each making different RNAs, and the one that makes messenger RNA needs a team of helper proteins to start.
College

RNA polymerase copies a template strand 3' to 5', building RNA 5' to 3' from ribonucleoside triphosphates without a primer. In E. coli the core enzyme (alpha2, beta, beta', omega) binds a sigma factor to form the holoenzyme; sigma 70 recognizes the -35 (consensus TTGACA) and -10 (TATAAT) elements, about 17 bp apart, melts the DNA into an open complex, and after rounds of abortive initiation the enzyme escapes the promoter. Termination is intrinsic (a GC-rich RNA hairpin followed by a U tract) or Rho-dependent. Eukaryotic nuclei have Pol I (large rRNA precursor), Pol II (pre-mRNAs, most snRNAs, miRNA precursors) and Pol III (tRNAs, 5S rRNA, U6). Pol II needs TFIID (with TBP), TFIIA, TFIIB, TFIIF, TFIIE and TFIIH to form a preinitiation complex; TFIIH opens the DNA and its CDK7 subunit phosphorylates Ser5 of the CTD heptad repeats. Pol II termination is coupled to cleavage and polyadenylation of the RNA.
Graduate

Initiation is the regulated step in both systems, and its intermediates are now seen structurally: closed complex, open complex with a melted bubble of roughly a turn of DNA, and an initial transcribing complex that scrunches downstream DNA into itself during abortive cycles until the RNA displaces the sigma region that blocks the RNA exit path. In eukaryotes TFIIH's XPB translocase drives promoter opening, and Ser5 phosphorylation of the CTD by CDK7 accompanies escape and recruitment of the capping enzyme. Two textbook pictures have been revised: in single-molecule work with purified E. coli enzyme, a substantial subpopulation of elongation complexes kept sigma 70; and in the Bacillus subtilis terminator atlas, Rho stimulated about 10% of intrinsic terminators, blurring the two classes.

For human Pol II, rapid depletion of CPSF73 in HCT116 cells caused widespread readthrough, and inhibiting the phosphatase PP1 extended readthrough after XRN2 loss, which the authors read as support for a combined model: slowing of the polymerase beyond the poly(A) site lets XRN2 catch it. An open question is how the relative weight of slowing and pursuit varies among genes and RNA classes, and what physically releases the polymerase from the DNA.
A mushroom toxin that sorts polymerases

Alpha-amanitin is a toxin made by the death cap mushroom and some of its relatives. In mammalian cells it blocks Pol II at low concentrations, Pol III only at much higher ones, and leaves Pol I largely untouched. That made it a laboratory tool long before anyone knew how it worked: add a chosen dose to isolated nuclei and see which RNAs stop being made. In 2002 the Kornberg laboratory reported a crystal structure of yeast Pol II with the toxin bound, in a pocket near the part of the enzyme that moves as the polymerase steps along the DNA.

— Bushnell, Cramer & Kornberg, Proc. Natl. Acad. Sci. USA 99:1218 (2002); Roeder & Rutter, Nature 224:234 (1969).
An antibiotic that plugs the exit

Rifampicin stops bacterial RNA polymerase but not the polymerases of the human nucleus. Resistance mutations had long mapped to the gene for the beta subunit. In 2001 Seth Darst, Alex Goldfarb and colleagues reported a structure of the core enzyme of Thermus aquaticus with rifampicin bound. The drug sat in the path that the new RNA takes out of the enzyme, more than 12 angstroms from the active site. The picture fit earlier biochemistry: the enzyme can begin an RNA in the drug's presence, but the chain is blocked after only two or three nucleotides.

— Campbell et al., Cell 104:901 (2001).
Sigma does not always let go

Many textbooks say sigma falls off as soon as the polymerase leaves the promoter. In 2016 Harden and colleagues watched single molecules of purified E. coli polymerase transcribing from a phage lambda promoter, with sigma 70 fluorescently labeled. A substantial subpopulation of elongating complexes kept sigma, and it left them slowly, at a measured rate of about 0.0038 per second. Release is real but not required at the moment of escape, at least in this purified system.

— Harden et al., Proc. Natl. Acad. Sci. USA 113:602 (2016).

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