DNA Replication and Gene Expression (AAMC 1B)
This page follows genetic information from the double helix to a folded protein: how DNA is copied and proofread, how damage is repaired, how a gene is transcribed and processed into mRNA, how ribosomes translate the code, and how cells decide which genes to switch on. On the MCAT these ideas show up in nearly every Bio/Biochem passage that involves a mutation, a knockout, a reporter gene or a drug that blocks a step of the central dogma.
The Central Dogma at a Glance
The central dogma says information flows DNA → RNA → protein. DNA is copied by replication, read into RNA by transcription, and decoded into a polypeptide by translation. Retroviruses add a reverse step (RNA → DNA, by reverse transcriptase), and some RNA viruses copy RNA directly (RNA-dependent RNA polymerase), but the core direction for cellular genes never changes: protein sequence is never copied back into nucleic acid.
Every nucleic acid polymerase reads the template 3′ → 5′ and builds the new strand 5′ → 3′, adding each nucleotide to a free 3′-OH. The energy comes from cleaving the incoming nucleoside triphosphate, releasing pyrophosphate (PPi), which is then hydrolyzed to make the reaction effectively irreversible. Ribosomes read mRNA 5′ → 3′ and build protein N-terminus → C-terminus.
DNA Replication
Replication is semiconservative: each daughter helix keeps one parental strand and gains one new strand. Meselson and Stahl showed this by growing bacteria in heavy 15N, switching to 14N, and finding a single intermediate-density band after one generation, then intermediate plus light bands after two.
Origins and the replication fork
Bacteria usually have one circular chromosome with a single origin (oriC), so replication runs bidirectionally until the two forks meet. Eukaryotic chromosomes are linear and much longer, so they use many origins at once. Each origin opens into a replication bubble with two forks moving in opposite directions.
| Protein | Job | Exam detail |
|---|---|---|
| Helicase | Unwinds the double helix by breaking hydrogen bonds between bases | Uses ATP; opens the fork |
| Single-strand binding proteins | Coat exposed single strands | Prevent re-annealing and protect from nucleases |
| Topoisomerase (gyrase in bacteria) | Relieves supercoiling ahead of the fork by cutting and rejoining DNA | Target of fluoroquinolone antibiotics and some anticancer drugs |
| Primase | Lays down a short RNA primer | DNA polymerase cannot start a chain from scratch; it needs a 3′-OH |
| DNA polymerase III (prokaryotes); δ and ε (eukaryotes) | Main synthesis of new DNA | Highly processive; 3′ → 5′ exonuclease proofreading |
| DNA polymerase I (prokaryotes) | Removes RNA primers and fills the gaps with DNA | Has 5′ → 3′ exonuclease activity; in eukaryotes RNase H and FEN1 do this |
| DNA ligase | Seals nicks in the sugar-phosphate backbone | Joins Okazaki fragments into a continuous strand |
| Telomerase | Extends chromosome ends using its own RNA template | A reverse transcriptase; active in germ cells, stem cells and most cancers |
Leading and lagging strands
Because new DNA can only grow 5′ → 3′, the two template strands are copied differently. The leading strand is made continuously toward the fork from one primer. The lagging strand is made away from the fork in short pieces called Okazaki fragments (roughly 1,000 to 2,000 nucleotides in bacteria and 100 to 200 in eukaryotes), each with its own RNA primer. The primers are later replaced with DNA and ligase joins the pieces.
Fidelity
Base pairing alone would give roughly one error per 104 to 105 nucleotides. Proofreading by the polymerase's 3′ → 5′ exonuclease lowers this about a hundredfold, and post-replication mismatch repair lowers it further, to roughly one error per 109 to 1010 nucleotides overall.
The end-replication problem and telomeres
On a linear chromosome, the final RNA primer at the very end of the lagging strand cannot be replaced with DNA, so each round of replication would shorten the chromosome. Telomeres are repetitive non-coding sequences (TTAGGG repeats in humans) that act as a buffer. Telomerase extends the 3′ overhang; somatic cells with little telomerase shorten their telomeres with each division, which contributes to replicative senescence.
DNA Repair
DNA is damaged constantly by spontaneous chemistry (deamination, depurination), reactive oxygen species, UV light and chemicals. Repair pathways are matched to the type of damage. Defects in these pathways raise mutation rates and cancer risk, which is why passages often pair a repair gene with a hereditary cancer syndrome.
| Pathway | Damage fixed | How it works | Linked condition |
|---|---|---|---|
| Mismatch repair | Wrong base pairs missed by proofreading | Recognizes the newly made strand (in E. coli, it is not yet methylated), cuts out a stretch and resynthesizes it | Lynch syndrome (hereditary nonpolyposis colorectal cancer) |
| Base excision repair | Single damaged or wrong bases (e.g., uracil from cytosine deamination) | A glycosylase removes the base, an endonuclease cuts the backbone, polymerase and ligase fill in | Rare syndromes; key for oxidative damage |
| Nucleotide excision repair | Bulky, helix-distorting lesions such as UV-induced thymine dimers | Excises an oligonucleotide around the lesion, then fills the gap | Xeroderma pigmentosum (extreme UV sensitivity) |
| Homologous recombination | Double-strand breaks, after S phase | Uses the sister chromatid as an accurate template | BRCA1 and BRCA2 mutations |
| Non-homologous end joining | Double-strand breaks, any phase | Ligates ends directly; fast but can delete or add bases | Also used in V(D)J recombination in lymphocytes |
Mutation types to name on sight
- Point mutations: silent (same amino acid, thanks to code degeneracy), missense (different amino acid), nonsense (creates a premature stop codon).
- Frameshift: insertion or deletion of a number of bases not divisible by 3, which shifts the reading frame downstream and usually creates an early stop.
- Chromosomal changes: deletions, duplications, inversions and translocations of larger segments.
Transcription
Transcription copies one strand of a gene into RNA. The strand that RNA polymerase reads is the template (antisense) strand; the RNA has the same sequence as the coding (sense) strand, with U in place of T. RNA polymerase does not need a primer and has no proofreading exonuclease, so transcription is less accurate than replication, but errors are not inherited.
Initiation, elongation, termination
- Initiation: in bacteria, the sigma factor helps RNA polymerase recognize promoter elements around −10 (Pribnow box, TATAAT) and −35. In eukaryotes, general transcription factors such as TFIID bind the TATA box (about 25 bases upstream) and recruit RNA polymerase II.
- Elongation: the polymerase unwinds a short stretch of DNA and adds ribonucleotides 5′ → 3′.
- Termination: bacteria use either a GC-rich hairpin followed by a run of U's (intrinsic) or the Rho helicase (Rho-dependent). Eukaryotic Pol II transcripts are cleaved after a polyadenylation signal (AAUAAA).
| Enzyme | Location | Main products |
|---|---|---|
| RNA polymerase I | Nucleolus | Large rRNAs (28S, 18S, 5.8S) |
| RNA polymerase II | Nucleoplasm | mRNA precursors (hnRNA), most snRNAs, miRNAs |
| RNA polymerase III | Nucleoplasm | tRNA, 5S rRNA |
RNA Processing in Eukaryotes
Bacterial mRNA is used as soon as it is made; transcription and translation are coupled in the cytoplasm. Eukaryotic primary transcripts are modified in the nucleus before export:
- 5′ cap: a 7-methylguanosine joined by an unusual 5′-5′ triphosphate bridge. It protects the mRNA from exonucleases and helps the ribosome find the message.
- Poly-A tail: about 200 adenines added to the 3′ end. It protects against degradation and helps export; tail length affects mRNA lifespan.
- Splicing: the spliceosome (snRNAs plus proteins, the snRNPs) removes introns and joins exons. The intron leaves as a lariat. Alternative splicing lets one gene encode several protein isoforms, which is a major reason humans have far more proteins than genes.
The Genetic Code and Translation
Features of the code
- Triplet: each codon is 3 nucleotides, giving 43 = 64 codons.
- Degenerate (redundant): 61 sense codons encode 20 amino acids, so most amino acids have more than one codon. Variation is usually in the third position, the wobble position, which can pair loosely with the tRNA anticodon.
- Unambiguous: each codon specifies only one amino acid.
- Start: AUG (methionine; N-formylmethionine in bacteria). Stops: UAA, UAG, UGA, which bind release factors rather than tRNAs.
- Nearly universal: shared across almost all life, with small exceptions such as mitochondrial codes.
Template: 3′-TAC GGA TTC-5′. Pair each base and flip the orientation label: mRNA 5′-AUG CCU AAG-3′. The coding strand reads 5′-ATG CCT AAG-3′, identical to the mRNA except T for U. Always write sequences 5′ → 3′ before you look up codons.
The players
- tRNA: cloverleaf-shaped adapter with an anticodon loop and a 3′ CCA end that carries the amino acid. Aminoacyl-tRNA synthetases attach the correct amino acid, using ATP; this "charging" step is where the code is really enforced.
- Ribosome: 70S in bacteria (30S + 50S) and 80S in eukaryotes (40S + 60S). It has three tRNA sites: A (aminoacyl, incoming), P (peptidyl, holds the growing chain) and E (exit). The peptidyl transferase activity is catalyzed by rRNA, making the ribosome a ribozyme.
Steps
- Initiation: the small subunit binds the mRNA (at the Shine-Dalgarno sequence in bacteria, or by scanning from the 5′ cap in eukaryotes), the initiator tRNA pairs with AUG in the P site, and the large subunit joins. Initiation factors and GTP are required.
- Elongation: a charged tRNA enters the A site (EF-Tu and GTP), a peptide bond forms as the chain transfers to the A-site amino acid, and the ribosome moves one codon (EF-G and GTP), shifting tRNAs from A to P to E.
- Termination: a stop codon in the A site binds a release factor; water attacks the ester bond and the polypeptide is released.
Energy bookkeeping: charging a tRNA costs 2 high-energy bonds (ATP → AMP), entry to the A site costs 1 GTP and translocation 1 GTP, so roughly 4 high-energy bonds per peptide bond.
After translation
Proteins may be folded with chaperones, cleaved (proinsulin to insulin), or modified by phosphorylation, glycosylation, acetylation, lipidation or ubiquitination. Proteins with an N-terminal signal sequence are made on ribosomes docked to the rough ER by the signal recognition particle and enter the secretory pathway.
Prokaryotic Gene Regulation: Operons
An operon is a cluster of genes under one promoter and one operator, transcribed as a single polycistronic mRNA. A repressor protein binds the operator and blocks RNA polymerase.
Genes lacZ (β-galactosidase), lacY (permease), lacA (transacetylase). No lactose: the LacI repressor binds the operator, so transcription is off. Lactose present: allolactose binds the repressor and releases it from the operator. Glucose low: cAMP rises, cAMP binds CAP (catabolite activator protein), and CAP-cAMP binds near the promoter to boost RNA polymerase binding. Maximal transcription needs lactose present and glucose absent. This glucose preference is called catabolite repression.
Encodes enzymes that make tryptophan. The repressor is made inactive; when tryptophan is abundant, it acts as a corepressor, binds the repressor and activates it, shutting the operon off. A second layer, attenuation, uses ribosome stalling on the leader sequence to decide whether a terminator hairpin forms, which is possible only because bacterial transcription and translation are coupled.
Eukaryotic Gene Regulation
Eukaryotic control happens at many levels, from chromatin to protein lifespan.
- Chromatin structure: DNA wraps around histone octamers to form nucleosomes. Histone acetylation (by HATs) neutralizes lysine positive charges, loosens chromatin (euchromatin) and generally increases transcription; deacetylation (HDACs) does the opposite. Tightly packed heterochromatin is largely silent.
- DNA methylation: methylation of cytosine in CpG islands near promoters usually silences genes. It underlies genomic imprinting and X-inactivation, and these epigenetic marks can be passed to daughter cells without any change in sequence.
- Transcription factors and enhancers: activators bind enhancers, which can sit thousands of bases away, upstream, downstream or in introns. DNA looping brings them to the promoter. Silencers bind repressors. Steroid hormone receptors are transcription factors that bind hormone response elements.
- RNA-level control: alternative splicing, mRNA stability (poly-A length, AU-rich elements), and RNA interference: microRNAs and siRNAs guide the RISC complex to block translation or cut target mRNA.
- Protein-level control: post-translational modification and degradation by the ubiquitin-proteasome system.
Links to Biotechnology
Most lab techniques reuse the enzymes on this page. PCR borrows replication (primers, a heat-stable DNA polymerase, dNTPs). Reverse transcriptase turns mRNA into cDNA for cloning or RT-PCR. Restriction enzymes and ligase build recombinant plasmids, and reporter genes placed behind a promoter or enhancer reveal when that regulatory element is active. CRISPR-Cas9 makes a targeted double-strand break and then relies on the cell's own NHEJ (to knock genes out) or homologous recombination (to insert a template). Details are on the biotechnology notes.
Worked MCQs
Five MCQs on the patterns the exam returns to most often. Read every explanation, including the ones you got right.
Q1. E. coli cells are moved to a medium that contains lactose but no glucose. Which statement best describes the lac operon in these cells?
With lactose present, allolactose binds the LacI repressor and removes it from the operator. With glucose absent, cAMP levels are high, so CAP-cAMP binds near the promoter and recruits RNA polymerase. Both conditions together give maximal transcription. Corepressors belong to repressible operons such as trp.
Q2. In E. coli, which enzyme removes the RNA primers from Okazaki fragments and replaces them with DNA?
DNA polymerase I has a 5′ to 3′ exonuclease that removes RNA primers and fills the gap with DNA. Polymerase III does bulk synthesis, primase makes the primers, and ligase only seals the remaining nick in the backbone.
Q3. A DNA template strand reads 3′-TACGGA-5′. What is the sequence of the mRNA transcribed from it?
RNA is antiparallel and complementary to the template, with U in place of T. Pairing 3′-TACGGA-5′ gives 5′-AUGCCU-3′, which begins with the start codon AUG. The option with T is the coding strand of DNA, not RNA.
Q4. A single base change converts the codon UAC (tyrosine) to UAA. This mutation is best classified as:
UAA is a stop codon, so translation ends early and a truncated protein results: a nonsense mutation. A single substitution cannot shift the reading frame, and the amino acid is not swapped for another one (missense) or kept the same (silent).
Q5. Patients with xeroderma pigmentosum develop skin cancers after minimal sun exposure. Which repair pathway is most likely defective?
UV light creates thymine (pyrimidine) dimers, bulky lesions that distort the helix. These are removed by nucleotide excision repair. Mismatch repair fixes replication errors, base excision repair fixes single damaged bases, and NHEJ fixes double-strand breaks.
Quick Recap
- Polymerases read 3′ → 5′ and build 5′ → 3′; DNA polymerase needs a primer, RNA polymerase does not.
- Replication is semiconservative; leading strand continuous, lagging strand in Okazaki fragments joined by ligase.
- Telomerase (a reverse transcriptase) solves the end-replication problem in germ, stem and cancer cells.
- Repair: mismatch (replication errors), BER (single bases), NER (thymine dimers), HR and NHEJ (double-strand breaks).
- Eukaryotic mRNA gets a 5′ cap, poly-A tail and splicing before export; alternative splicing gives isoforms.
- 64 codons, 61 sense, 3 stops; AUG start; code is degenerate but unambiguous.
- lac operon: on with lactose and without glucose (inducible). trp operon: off with tryptophan (repressible).
- Acetylation opens chromatin, DNA methylation silences; enhancers act at a distance; miRNAs silence mRNAs.