AP Syllabus focus:
‘RNA polymerase synthesizes RNA in the 5? to 3? direction while reading the DNA template in the 3? to 5? direction.’
Transcription directionality is a core rule that links nucleic-acid structure to gene expression. Understanding how RNA polymerase reads one strand and builds RNA explains gene orientation, RNA sequence prediction, and many common student errors.
Core idea: direction is built into nucleic acids
DNA and RNA strands have polarity because nucleotides are linked in a consistent orientation along the sugar-phosphate backbone.
5′ end and 3′ end: The 5′ end has a phosphate on the sugar’s 5′ carbon; the 3′ end has a hydroxyl (3′ OH) on the 3′ carbon, which is the site where new nucleotides are added.
Because strands have polarity, any polymerase must move along a template strand in one direction while building the complementary strand in the opposite (antiparallel) direction.
RNA polymerase directionality (what to memorise)
The syllabus rule
RNA polymerase synthesises RNA 5′ → 3′
RNA polymerase reads the DNA template 3′ → 5′
This is not a convention; it reflects the chemistry of chain growth: adding an incoming ribonucleotide requires a free 3′ OH on the growing RNA.
What “synthesises 5′ → 3′” means
New ribonucleotides are added only to the 3′ end of the growing RNA strand.
Therefore, the RNA’s 5′ end is produced first, and the 3′ end is produced last.
The RNA sequence is written 5′ → 3′ by convention, matching how it is extended.
What “reads the template 3′ → 5′” means
The template strand must be oriented so RNA polymerase can move along it in the 3′ → 5′ direction.
Each DNA base on the template dictates the complementary RNA base that will be added:
DNA A → RNA U
DNA T → RNA A
DNA C → RNA G
DNA G → RNA C
Template strand vs coding strand (directionality avoids confusion)
In any transcription unit, only one DNA strand is used as the template for RNA synthesis; the other strand is not “ignored,” but it is not the strand being read by RNA polymerase for that RNA.
Template strand: The DNA strand read by RNA polymerase (3′ → 5′) to assemble a complementary RNA strand (5′ → 3′).
A useful consequence of directionality:
This schematic shows the transcription bubble with RNA polymerase adding ribonucleotides to the RNA 3′ end, so the transcript elongates 5′ → 3′ while the DNA template is read antiparallel. It also highlights the relationship between strands: the RNA sequence matches the coding (non-template) strand except that U replaces T, while remaining complementary to the template strand. Source
The RNA sequence (5′ → 3′) matches the non-template (coding) strand sequence written 5′ → 3′, except RNA has U where DNA has T.
This minimalist cartoon contrasts the coding (non-template) and template strands and visually reinforces antiparallel geometry during transcription. It is especially helpful for sequence translation practice: read the template 3′ → 5′, write RNA 5′ → 3′, and remember that the coding strand matches the RNA (with T → U). Source
The RNA is complementary and antiparallel to the template strand.
Why directionality matters for gene layout and interpretation
Genes have an orientation on DNA
Because RNA polymerase must read 3′ → 5′ on the template, a gene’s position is not just “where it is,” but also which direction it is transcribed.
RNA polymerase can bind and move along DNA in either physical direction, but in each case it must traverse the chosen template strand in the 3′ → 5′ direction to produce an RNA strand that grows 5′ → 3′. The figure emphasizes that promoter orientation fixes polymerase orientation, which in turn determines which DNA strand functions as the template for that gene. Source
Two genes can occupy the same chromosome region but be transcribed in opposite directions if they use opposite template strands.
Common directionality checkpoints for students
If you are given a DNA template written 3′ → 5′, the RNA you produce should be written 5′ → 3′ and be complementary.
If you are given a DNA coding strand written 5′ → 3′, the RNA will match it 5′ → 3′ with T → U substitutions.
If your RNA appears to be growing from its 3′ end toward its 5′ end, the direction has been reversed; RNA chain growth does not proceed 3′ → 5′.
Directionality constrains what RNA polymerase can do
RNA polymerase cannot extend from the RNA 5′ end because there is no chemical mechanism to add nucleotides there during polymerisation.
Any pause, proofreading-like correction, or restart must still preserve the rule: the next nucleotide is added to the RNA 3′ end.
FAQ
Polymerisation relies on the 3′ OH of the growing RNA attacking the phosphate of an incoming nucleoside triphosphate.
This arrangement is energetically favourable because the incoming NTP carries the reactive triphosphate group.
They can map RNA back to the genome using strand-specific sequencing methods.
Alternatively, hybridisation approaches use labelled probes designed to bind only one strand orientation, revealing which strand is complementary to the RNA.
RNA polymerase can backtrack along DNA during pauses.
Directionality is not violated because when synthesis resumes, nucleotides are still added to the RNA 3′ end, maintaining overall 5′ → 3′ synthesis.
Many regulatory elements are orientation-dependent because they must recruit polymerase in the correct direction.
If the transcription complex assembles facing the opposite way, the wrong strand would be positioned as template for that transcription unit.
You can invert the DNA region containing the transcription signals that set polymerase orientation.
Practically, researchers often clone the target sequence into a vector in the reverse orientation relative to a unidirectional promoter to reverse the RNA produced.
Practice Questions
State the direction in which RNA polymerase (i) reads the DNA template strand and (ii) synthesises the RNA strand. (2 marks)
Reads template strand 3′ → 5′ (1)
Synthesises RNA 5′ → 3′ (1)
Explain how transcription directionality determines (a) which DNA strand is used as the template and (b) the relationship between the RNA sequence and the two DNA strands. (5 marks)
RNA polymerase synthesises RNA 5′ → 3′ by adding nucleotides to the 3′ end (1)
Therefore it must read the DNA template 3′ → 5′ (1)
Only the DNA strand oriented appropriately can serve as the template strand for that transcription unit (1)
RNA is complementary and antiparallel to the template strand (1)
RNA matches the coding (non-template) strand sequence (when both written 5′ → 3′) except U replaces T (1)
