Transcription &
RNA Processing
Master how DNA is read by RNA polymerase, the stages of transcription, and the post-transcriptional modifications that produce mature mRNA.
MCAT Biological Foundations
What Is Transcription?
Transcription is the process by which the information encoded in a DNA template strand is copied into a complementary messenger RNA (mRNA) molecule. It is the first step in gene expression: converting a gene's nucleotide sequence into a functional product.
Key Molecular Players
RNA Polymerase
Enzyme that reads DNA \(3' \rightarrow 5'\) and synthesizes RNA \(5' \rightarrow 3'\). Eukaryotes use Pol I, II, III.
Template Strand
The \(3' \rightarrow 5'\) DNA strand that RNA polymerase reads. Also called the antisense strand.
Coding Strand
The \(5' \rightarrow 3'\) DNA strand identical in sequence to the mRNA (except \(T \rightarrow U\)). Also called the sense strand.
Eukaryotic RNA Polymerases
| RNA Pol I | RNA Pol II | RNA Pol III | |
|---|---|---|---|
| Product | rRNA (28S, 18S, 5.8S) | mRNA, snRNA | tRNA, 5S rRNA |
| Location | Nucleolus | Nucleoplasm | Nucleoplasm |
| Sensitivity | Not inhibited | Inhibited by α-amanitin | High-dose α-amanitin |
Three Stages of Transcription
Initiation: Promoter Recognition
1
Transcription factors (e.g., TFIID) bind the TATA box (\(\sim 25 \text{ bp}\) upstream of the start site) in eukaryotes.
2
RNA Polymerase II is recruited to the promoter, forming the pre-initiation complex (PIC).
3
The DNA double helix unwinds (\(\sim 14 \text{ bp}\)), forming an open complex or transcription bubble.
4
RNA Pol begins synthesizing RNA from the \(+1\) start site, reading the template strand \(3' \rightarrow 5'\).
Elongation: Building the RNA
During elongation, RNA polymerase moves along the template strand, adding ribonucleotides (A, U, G, C) to the growing mRNA chain in the \(5' \rightarrow 3'\) direction. The transcription bubble moves with the polymerase.
Base Pairing
A (template) \( \rightarrow \) U (RNA), T \( \rightarrow \) A, G \( \rightarrow \) C, C \( \rightarrow \) G
Bond Formed
Phosphodiester bonds link nucleotides. Energy from NTP hydrolysis (releasing PPi).
Termination: Releasing the Transcript
Prokaryotic (Rho-independent)
A GC-rich hairpin loop followed by a poly-U tail forms in the RNA, causing the polymerase to stall and dissociate.
Prokaryotic (Rho-dependent)
The Rho (ρ) protein, a helicase, catches up to the stalled polymerase and unwinds the RNA:DNA hybrid.
Eukaryotic
Cleavage at the polyadenylation signal (AAUAAA) releases the pre-mRNA. Pol II continues briefly then detaches.
Prokaryotic vs Eukaryotic Transcription
| Prokaryotes | Eukaryotes | |
|---|---|---|
| RNA Polymerase | One type (core + σ) | Three types (Pol I, II, III) |
| Promoter | -10 (Pribnow), -35 | TATA box, enhancers |
| Coupling | Coupled (transcription + translation simultaneous) | Uncoupled (RNA processed in nucleus first) |
| mRNA Processing | None (polycistronic) | 5' cap, poly-A tail, splicing |
| Introns | Rare | Common: removed by splicing |
Post-Transcriptional RNA Processing
In eukaryotes, the pre-mRNA undergoes three critical modifications before leaving the nucleus as mature mRNA:
5' Cap and Poly-A Tail
5' Cap
A 7-methylguanosine (m7G) is added to the 5' end via a \(5' \rightarrow 5'\) triphosphate linkage. Protects from exonucleases, aids ribosome recognition, and facilitates nuclear export.
3' Poly-A Tail
Poly-A polymerase adds ~200 adenine nucleotides after cleavage at the AAUAAA signal. Protects from degradation, aids nuclear export, and enhances translation efficiency.
RNA Splicing: Introns Out, Exons Together
1
snRNPs (small nuclear ribonucleoproteins) recognize consensus sequences at intron-exon boundaries: GU at the 5' splice site and AG at the 3' splice site.
2
Five snRNPs (U1, U2, U4, U5, U6) assemble into the spliceosome, a large ribozyme complex.
3
The intron forms a lariat loop structure via a \(2'\text{--}5'\) phosphodiester bond at the branch point adenine.
4
The lariat is excised and degraded. Flanking exons are ligated together to form continuous mRNA.
Alternative Splicing: One Gene, Many Proteins
How to Approach Transcription Problems
1
Identify the template strand. RNA Pol reads \(3' \rightarrow 5'\). The strand given as \(3' \rightarrow 5'\) is the template.
2
Write complementary bases. \(A \rightarrow U\), \(T \rightarrow A\), \(G \rightarrow C\), \(C \rightarrow G\). Remember: RNA uses uracil (U) instead of thymine (T).
3
Write mRNA \(5' \rightarrow 3'\). This matches the coding strand sequence (with U replacing T).
4
Verify direction. mRNA is always synthesized and read \(5' \rightarrow 3'\). Double-check your strand orientation.
Example 1: Writing the mRNA
Given: Template strand: \(3'\text{-TACGGATCCA-}5'\)
Example 2: Mutation Effects
Scenario: A point mutation changes the template strand sequence from \(3'\text{-TAC-}5'\) to \(3'\text{-TTC-}5'\) at the start of a gene.
Normal
Template: \(3'\text{-TAC-}5' \rightarrow\) mRNA: \(5'\text{-AUG-}3' \rightarrow\) Met (start codon, translation begins)
Mutant
Template: \(3'\text{-TTC-}5' \rightarrow\) mRNA: \(5'\text{-AAG-}3' \rightarrow\) Lys (no start codon, translation cannot initiate normally)
Common Mistakes
✕ Confusing Template and Coding
The template strand (\(3' \rightarrow 5'\)) is read by RNA Pol. The coding strand has the same sequence as mRNA (with T instead of U).
✕ Forgetting U Replaces T
RNA uses uracil, not thymine. Writing T in an mRNA sequence is always wrong.
✕ Wrong Strand Direction
mRNA is synthesized \(5' \rightarrow 3'\). If you write it \(3' \rightarrow 5'\), your codon reading frame will be reversed.
Summary: RNA Processing Steps
| Modification | What Happens | Function |
|---|---|---|
| 5' Capping | 7-methylguanosine added via \(5'\text{--}5'\) linkage | Ribosome recognition, stability, nuclear export |
| 3' Polyadenylation | ~200 A residues added after AAUAAA signal | Stability, export, translation efficiency |
| Splicing | Introns removed by spliceosome (snRNPs) | Joins exons, enables alternative splicing |
| Alternative Splicing | Different exon combinations included | Proteome diversity from single gene |
Transcription Regulation Highlights
1
Enhancers and silencers: DNA sequences (can be thousands of bp away) that increase or decrease transcription when bound by transcription factors.
2
Chromatin remodeling: Histone acetylation loosens chromatin (euchromatin) \( \rightarrow \) more transcription. Deacetylation tightens it (heterochromatin) \( \rightarrow \) less transcription.
3
DNA methylation: Adding methyl groups to CpG islands in the promoter typically silences gene expression.
4
Operons (prokaryotes): The lac operon and trp operon use repressors/activators to control transcription of gene clusters.
Let's Review
You've learned the key stages of transcription, the molecular machinery involved, and how pre-mRNA is processed into mature mRNA. Time to test your understanding with 5 assessment questions.
Question 1 of 5
RNA polymerase reads the template strand in which direction?
RNA polymerase always reads the template strand \(3' \rightarrow 5'\) and synthesizes the new mRNA strand \(5' \rightarrow 3'\). This is a fundamental rule of transcription.
Question 2 of 5
The 5' cap of eukaryotic mRNA is composed of:
The 5' cap is a 7-methylguanosine (m7G) added through an unusual \(5' \rightarrow 5'\) triphosphate linkage. It protects from exonuclease degradation and is recognized by the ribosome during translation initiation.
Question 3 of 5
If the template strand reads \(3'\text{-AATCGC-}5'\), what is the resulting mRNA sequence?
Complement each base (\(A \rightarrow U\), \(T \rightarrow A\), \(C \rightarrow G\), \(G \rightarrow C\)) and write the mRNA \(5' \rightarrow 3'\) (antiparallel to the \(3' \rightarrow 5'\) template). A\(\rightarrow\)U, A\(\rightarrow\)U, T\(\rightarrow\)A, C\(\rightarrow\)G, G\(\rightarrow\)C, C\(\rightarrow\)G gives \(5'\text{-UUAGCG-}3'\). Choice B uses T instead of U. Choice C has the correct sequence but reversed directionality. Choice D is the reverse complement, a common error from reading the template in the wrong direction.
Question 4 of 5
Which component of the spliceosome provides catalytic activity during intron removal?
The spliceosome is a ribozyme: the catalytic activity comes from the snRNA molecules (especially U2 and U6 snRNAs), not from the protein components. This is key evidence for the RNA World hypothesis.
Question 5 of 5
A researcher treats eukaryotic cells with α-amanitin. Which RNA species would be most directly affected?
α-amanitin is a potent inhibitor of RNA Polymerase II, which synthesizes mRNA (and some snRNAs). RNA Pol I (rRNA) is insensitive, and RNA Pol III (tRNA, 5S rRNA) requires much higher concentrations to be inhibited.