Template Strand

The template strand (also designated the non-coding strand, transcribed strand, antisense strand, minus (−) strand, or Crick strand) is the polynucleotide strand of a double-stranded DNA duplex that is read by a DNA-dependent RNA polymerase during transcription—or by a DNA polymerase during DNA replication—to direct the synthesis of a complementary nucleic acid polymer via Watson–Crick hydrogen bonding. Because cellular nucleic acid polymerases catalyze phosphodiester bond formation in the 5′ → 3′ direction (via nucleophilic attack of the nascent chain's 3′-OH on the α-phosphate of an incoming nucleoside triphosphate) and pair strands in an antiparallel orientation, the polymerase translocates along the template strand in the 3′ → 5′ direction. Consequently, during transcription of a gene, the nascent mRNA transcript (5′ → 3′) is complementary and antiparallel to the template strand (3′ → 5′), and matches the sequence and 5′ → 3′ polarity of the opposite, non-transcribed partner strand—termed the coding strand (sense / non-template / plus (+) strand)—except that uracil (U) replaces thymine (T) at every position.

Resolving Synonymous Nomenclature Across Molecular Biology

Because the two strands of a B-form DNA double helix are antiparallel (5′ → 3′ paired with 3′ → 5′) and complementary (A=T, G≡C), molecular biologists refer to the two strands of a transcribed locus using paired functional terms depending on whether the reference point is enzymatic catalysis or bioinformatic sequence annotation:

By international bioinformatic convention (GenBank, Ensembl, UCSC Genome Browser), when the sequence of a single gene is written as a single line of text, it is reported as the 5′ → 3′ coding (non-template) strand, allowing researchers to read upstream promoter motifs, the 5′-ATG-3′ translation start codon, exon–intron splice junctions (5′-GT ... AG-3′), and triplet codons directly from left to right.

`` Promoter (+1 TSS) ──────────────────► Direction of Pol II Translocation Coding (Non-Template, +) Strand: 5'──TATAAT ... ──ATG CCG AAT GCT TGA──3' (Displaced ssDNA loop) x x x x x Template (Antisense, −) Strand: 3'──ATATTA ... ──TAC GGC TTA CGA ACT──5' (Read 3' → 5' by Pol II) ||| ||| ||| ||| ||| (8–9 bp RNA-DNA hybrid) Nascent mRNA Transcript: 5'──AUG CCG AAU GCU UGA──3' (Synthesized 5' → 3') ``

Mechanics of the 12–14 bp Transcription Bubble and 8–9 bp Hybrid

X-ray crystallographic and cryo-EM structures of bacterial RNA polymerase and eukaryotic RNA Polymerase II (resolved by Roger Kornberg and colleagues) reveal how the enzyme separates and tracks the template strand:

1. Promoter Melting (12–14 bp Transcription Bubble): Upon binding the core promoter, σ-factor aromatic residues in bacteria—or the ATP-dependent XPB translocase subunit of TFIIH in eukaryotic Pol II initiation—unwind 12 to 14 base pairs of duplex DNA around the +1 transcription start site (TSS).
2. Strand Partitioning by the Bridge Helix and Fork Loops: As downstream duplex DNA enters the main cleft of Pol II and bends ~90° over the bridge helix, the non-template (coding) strand is guided across the outer surface of the clamp, held apart by fork loop 2. Simultaneously, the template strand threads into the catalytic center, where the +1 templating base flips opposite active-site Mg²⁺ Ion A and Ion B coordinated by an invariant DFDGD aspartate motif.
3. The 8–9 bp RNA–DNA Hybrid and Lid Separation: Incoming ribonucleoside triphosphates (ATP, UTP, GTP, CTP) enter via the secondary pore and base-pair with the template strand, maintaining a 8 to 9 base-pair RNA–DNA A-form hybrid duplex inside the active cleft. At the upstream boundary of this hybrid, the rudder and lid loop peel the 5′ end of the nascent RNA away from the DNA template strand, routing the transcript out the RNA exit channel while the template strand re-anneals with the coding strand to restore the B-DNA helix.

Promoter Orientation and Chromosome-Wide Strand Choice

Template versus coding identity is defined per gene (per transcription unit) rather than across an entire chromosome. Which of the two chromosomal strands serves as the template for a given gene is determined by the asymmetric orientation of its promoter sequence:

• In Bacteria, the σ⁷⁰ subunit recognizes two asymmetric hexamers on the coding strand: the −35 element (5′-TTGACA-3′) and the −10 Pribnow box (5′-TATAAT-3′), orienting RNA polymerase in a single direction and selecting the opposite 3′ → 5′ strand as the template.
• In Eukaryotes, the TATA box (5′-TATAAA-3′), Initiator (Inr), and TFIIB recognition elements (BRE) orient the pre-initiation complex, while the U1 snRNP–PAS polyadenylation axis suppresses upstream antisense elongation from bidirectional CpG-island promoters.

Along any bacterial or eukaryotic chromosome, roughly half of all genes use the top reference strand as their template and half use the bottom strand, arranged in tandem (──► ──►), divergent (◄── ──►), or convergent (──► ◄──) orientations. In overlapping antisense loci, the same DNA segment serves simultaneously as the template strand for one gene and the coding strand for another.

`` Chromosome Duplex: Top Strand: 5'────[Gene 1 Coding]────► ◄────[Gene 2 Template]────3' Bottom Strand: 3'────[Gene 1 Template]──► ◄────[Gene 2 Coding]──────5' (Gene 1 transcribed (Gene 2 transcribed left-to-right ──►) ◄── right-to-left) ``

Template Strands in DNA Replication and Transcription-Coupled Repair (TC-NER)

During semi-conservative DNA replication, both parental DNA strands serve simultaneously as template strands at the replication fork. The parental strand oriented 3′ → 5′ toward the advancing fork acts as the leading-strand template, copied continuously (5′ → 3′) by DNA polymerase ε. The opposite parental strand oriented 5′ → 3′ toward the fork acts as the lagging-strand template, copied discontinuously (5′ → 3′ away from the fork) by Pol α-primase and Pol δ into 100–200 nt Okazaki fragments matured by FEN1 and DNA Ligase I.

In transcribed genes, bulky DNA lesions—such as UV cyclobutane pyrimidine dimers (CPDs) or cisplatin crosslinks—on the coding (non-template) strand do not block transcription and are repaired slowly by Global Genome Nucleotide Excision Repair (GG-NER) via XPC–RAD23B. Conversely, when a bulky lesion resides on the template (transcribed) strand, it stalls elongating RNA Polymerase II in the active cleft. Stalled Pol II recruits the Cockayne Syndrome B (CSB / ERCC6) ATPase and Cockayne Syndrome A (CSA / ERCC8) complex, triggering Transcription-Coupled Nucleotide Excision Repair (TC-NER) (discovered by Philip Hanawalt and Isabel Mellon in 1987), which recruits TFIIH, XPA, XPG, and XPF–ERCC1 to excise a 24–32 nt patch around the lesion. Because TC-NER repairs the template strand 5 to 10 times faster than GG-NER repairs the coding strand, active genes accumulate fewer UV and tobacco mutations on their template strand than on their coding strand.

Why do sequence databases like GenBank list the coding strand instead of the template strand that RNA polymerase reads?

By convention, all nucleic acid sequences are written in the 5′ → 3′ direction from left to right—the direction in which mRNA is synthesized by RNA polymerase and translated by the ribosome (N-terminus → C-terminus). Because the coding (non-template) strand runs 5′ → 3′ in the same polarity as the mRNA transcript and shares the same nucleotide sequence (with T in place of U), publishing the coding strand allows direct visual inspection of open reading frames, ATG start codons, triplet codons, and GT–AG splice boundaries from left to right.

Can the same strand of a chromosome be the template strand for one gene and the coding strand for another?

Yes—and this occurs thousands of times along every eukaryotic and bacterial chromosome. Because DNA is a double helix with genes distributed roughly equally across both orientations, moving along a single uninterrupted chromosomal strand (5′ → 3′ from telomere to telomere) encounters genes pointing in both directions. Wherever a gene's promoter directs transcription from left to right, the bottom (3′ → 5′) strand acts as the template strand and the top (5′ → 3′) strand acts as the coding strand; wherever a neighboring gene's promoter directs transcription from right to left, their roles invert.

How does strand-specific RNA-seq distinguish transcripts made from the two opposite strands of DNA?

Standard first-generation RNA-seq converted mRNA into double-stranded cDNA before ligating symmetric sequencing adapters, erasing information about which of the two cDNA strands matched the original RNA transcript. Strand-specific (directional) RNA-seq (most commonly the dUTP second-strand marking method) preserves template identity: during first-strand cDNA synthesis by reverse transcriptase, standard dNTPs (dATP, dTTP, dGTP, dCTP) are used to copy the RNA template; during second-strand synthesis, dUTP replaces dTTP, incorporating uracil into the second strand. Prior to PCR amplification, treatment with Uracil-DNA Glycosylase (UDG) degrades the uracil-containing second strand, ensuring that sequencing reads map to the original genomic template strand.

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