Biochemistry chapters
DNA replication and RNA synthesis
The structure of DNA and RNA, how DNA is copied (semi-conservative replication, the enzymes at the replication fork, telomerase) and how genes are transcribed into RNA (RNA polymerases, promoters, mRNA processing: cap, tail and splicing), with the drugs and toxins that block each step.
- DNA is a double helix of two antiparallel strands held by hydrogen bonds (A=T two bonds, G≡C three bonds; so GC-rich DNA melts at higher temperature); Chargaff's rule: A = T and G = C; in RNA, uracil replaces thymine and the sugar is ribose.
- Replication is semi-conservative and runs 5′→3′: helicase unwinds, topoisomerases relieve supercoiling (quinolones block bacterial gyrase), primase lays RNA primers, DNA polymerase extends them (continuous leading strand, Okazaki fragments on the lagging strand), and ligase seals the gaps; telomerase (a reverse transcriptase) maintains chromosome ends.
- Transcription: RNA polymerase II makes mRNA (blocked by α-amanitin of death-cap mushrooms), I makes rRNA, III makes tRNA; the pre-mRNA gets a 5′ cap, a poly-A tail and splicing (introns removed by snRNPs) before leaving the nucleus.
On this page
Structure of nucleic acids
Nucleotide = nitrogenous base + sugar + phosphate. A nucleoside is base + sugar only.
| Base | Type | Rings | In |
|---|---|---|---|
| Adenine (A) | Purine | Two | DNA and RNA |
| Guanine (G) | Purine | Two | DNA and RNA |
| Cytosine (C) | Pyrimidine | One | DNA and RNA |
| Thymine (T) | Pyrimidine | One | DNA only |
| Uracil (U) | Pyrimidine | One | RNA only |
"Pure As Gold": purines are A and G. "CUT the py": C, U, T are pyrimidines.
DNA
- Double helix (Watson and Crick): two antiparallel strands (one 5′→3′, the other 3′→5′) with a deoxyribose-phosphate backbone linked by phosphodiester bonds.
- Base pairing: A with T (two hydrogen bonds), G with C (three hydrogen bonds). DNA rich in G-C needs a higher temperature to separate (melt).
- Chargaff's rule: in double-stranded DNA, A = T and G = C, so purines = pyrimidines.
- Packaging: DNA wraps around histone octamers (H2A, H2B, H3, H4) to form nucleosomes ("beads on a string"); H1 links them. Histones are positively charged (rich in lysine and arginine). Heterochromatin is condensed and inactive; euchromatin is open and active. Histone acetylation loosens chromatin (more transcription); DNA methylation silences genes.
In double-stranded DNA: A = T and G = C, so A + G = 50%
A sample has 20% adenine:
T = 20% (pairs with A)
G + C = 100 − (20 + 20) = 60%
G = C = 60 ÷ 2 = 30% each
Check: purines A + G = 20 + 30 = 50% (matches)
(This does not apply to single-stranded DNA or RNA.)
RNA
- Usually single-stranded; sugar is ribose; uracil replaces thymine.
- Types: mRNA (carries the code), tRNA (brings amino acids; cloverleaf; anticodon), rRNA (most abundant; part of ribosomes), small nuclear RNA (snRNA: splicing), microRNA (gene silencing).
DNA replication
Semi-conservative: each new double helix has one old and one new strand (shown by Meselson and Stahl). It happens during S phase.
The replication fork
- Origin of replication: prokaryotes have one; eukaryotes have many (faster copying of large genomes).
- Helicase unwinds the double helix (uses ATP).
- Single-strand binding proteins keep strands apart.
- Topoisomerases relieve the supercoiling ahead of the fork:
- Type I cuts one strand (blocked by irinotecan, topotecan).
- Type II cuts both strands (bacterial DNA gyrase, blocked by fluoroquinolones; human topoisomerase II blocked by etoposide, doxorubicin).
- Primase makes a short RNA primer (DNA polymerase cannot start a chain on its own).
- DNA polymerase adds nucleotides to the 3′ end, so synthesis is always 5′ → 3′.
- Leading strand: made continuously towards the fork.
- Lagging strand: made discontinuously as Okazaki fragments away from the fork.
- RNA primers are removed and replaced with DNA (DNA polymerase I in prokaryotes, which has 5′→3′ exonuclease activity; RNase H and pol δ in eukaryotes).
- DNA ligase joins the fragments (seals nicks).
Proofreading: DNA polymerases have 3′ → 5′ exonuclease activity to remove wrong bases, giving very high accuracy.
DNA polymerases
| Prokaryotic | Role | Eukaryotic | Role |
|---|---|---|---|
| Pol III | Main replicating enzyme (both strands) | Pol α | Primase activity; starts strands |
| Pol I | Removes RNA primers (5′→3′ exonuclease) and fills gaps; repair | Pol δ | Lagging strand (and leading in some models) |
| Pol II | Repair | Pol ε | Leading strand |
| (none) | (none) | Pol γ | Mitochondrial DNA |
| (none) | (none) | Pol β | DNA repair |
Telomeres and telomerase
The lagging strand cannot be completed at the very end of a linear chromosome, so chromosomes shorten each division. Telomeres are repeated sequences (TTAGGG) at chromosome ends. Telomerase is a reverse transcriptase carrying its own RNA template that adds these repeats. It is active in germ cells, stem cells and most cancer cells (allowing unlimited division), but not in most adult cells (ageing).
Reverse transcriptase
Makes DNA from an RNA template: used by retroviruses (HIV) and hepatitis B; blocked by NRTIs (zidovudine, tenofovir) and NNRTIs. Used in the laboratory for RT-PCR.
Drugs that block DNA synthesis
| Drug | Target |
|---|---|
| Fluoroquinolones | Bacterial DNA gyrase (topoisomerase II) and topoisomerase IV |
| Etoposide, doxorubicin | Human topoisomerase II |
| Irinotecan, topotecan | Topoisomerase I |
| Aciclovir, ganciclovir | Viral DNA polymerase |
| Zidovudine, tenofovir | Reverse transcriptase |
| Cytarabine | DNA polymerase (chain termination) |
| Methotrexate, 5-FU, hydroxyurea | Nucleotide synthesis (indirectly) |
Transcription (RNA synthesis)
RNA polymerase reads the template (antisense) strand 3′→5′ and makes RNA 5′→3′, which matches the coding (sense) strand (with U for T). It needs no primer.
RNA polymerases
| Organism | Polymerase | Product | Inhibitor |
|---|---|---|---|
| Prokaryotes | One RNA polymerase (σ factor recognises the promoter) | All RNA | Rifampicin (binds β subunit) |
| Eukaryotes | RNA polymerase I | rRNA (in the nucleolus) | (none commonly tested) |
| Eukaryotes | RNA polymerase II | mRNA (and some snRNA, microRNA) | α-Amanitin (death-cap mushroom Amanita phalloides: liver failure) |
| Eukaryotes | RNA polymerase III | tRNA, 5S rRNA | (high-dose α-amanitin) |
Polymerases I, II, III make r, m, t RNA, in that order ("rmt": rich men travel).
Actinomycin D intercalates DNA and blocks transcription in both prokaryotes and eukaryotes.
Promoters and regulation
- Promoter: the DNA site where RNA polymerase binds, upstream of the gene. Eukaryotes: TATA box (Hogness box) about 25 bases upstream, CAAT box. Prokaryotes: Pribnow box (TATAAT, −10) and −35 sequence.
- General transcription factors (TFIID binds the TATA box) are needed by RNA polymerase II.
- Enhancers: increase transcription; can act from far away, upstream or downstream; silencers reduce it.
- Prokaryotic operons: the lac operon is switched on by lactose (inducer removes the repressor) when glucose is low (cAMP-CAP).
Processing of eukaryotic mRNA (in the nucleus)
- 5′ cap: 7-methylguanosine; protects mRNA and helps ribosome binding.
- 3′ poly-A tail: about 200 adenines added after the AAUAAA signal; stability and export.
- Splicing: removal of introns (non-coding) and joining of exons by the spliceosome (snRNPs). Antibodies against snRNPs (anti-Smith) are specific for SLE. Alternative splicing lets one gene make several proteins. Splicing mutations cause some β-thalassaemias.
Prokaryotic mRNA is not processed: transcription and translation happen together.
Expect: purines vs pyrimidines, hydrogen bonds in A-T and G-C, Chargaff's rule calculations, semi-conservative replication, the enzymes of the replication fork (helicase, topoisomerase, primase, polymerases, ligase), Okazaki fragments, prokaryotic pol I vs III, telomerase, reverse transcriptase, RNA polymerase types and inhibitors (α-amanitin, rifampicin), promoters (TATA box), and mRNA processing (cap, tail, splicing, anti-Smith).
Quick revision
- Purines A, G; pyrimidines C, T, U.
- A=T 2 bonds; G≡C 3 bonds.
- Chargaff: A = T, G = C (double-stranded DNA).
- Replication: semi-conservative, 5′→3′, leading and lagging (Okazaki) strands.
- Helicase, topoisomerase (quinolones), primase, polymerase III/I, ligase.
- Telomerase: reverse transcriptase; stem and cancer cells.
- RNA pol I rRNA, II mRNA (α-amanitin), III tRNA; bacterial pol: rifampicin.
- mRNA processing: 5′ cap, poly-A tail, splicing (snRNPs).
Written by The House Job from standard biochemistry references (Lippincott Illustrated Reviews: Biochemistry).
FAQ
How many hydrogen bonds join guanine and cytosine?
Three; adenine and thymine are joined by two.
What are Okazaki fragments?
Short pieces of DNA made on the lagging strand during replication, later joined by DNA ligase.
Which RNA polymerase makes messenger RNA?
RNA polymerase II, which is inhibited by α-amanitin.
What does telomerase do?
It adds repeat sequences to chromosome ends using its own RNA template, preventing shortening; it is active in stem cells and most cancers.