How a Cell Copies Its DNA and Reads Its Genes

Before a cell divides it must copy its DNA exactly, and to make proteins it must read genes into RNA and then translate that RNA into amino acids. This flow — DNA to RNA to protein — is known as the central dogma.

This part covers semiconservative replication, the replication machinery, transcription, and the genetic code with translation.

In semiconservative replication each parent strand acts as a template, so every daughter molecule has one old strand and one new strand; Meselson and Stahl proved this by growing E. coli on heavy nitrogen, moving it to light nitrogen, and tracking the density of its DNA over generations.

Meselson-Stahl experiment:

- E. coli was grown for many generations in medium containing **${}^{15}$N, heavy nitrogen, so all its DNA became heavy
- The cells were moved to ${}^{14}$N medium and allowed to divide
- DNA was separated by density-gradient centrifugation in caesium chloride

Results:

- After one generation, about 20 minutes, all DNA had hybrid density
- After two generations, there were equal amounts of hybrid and light DNA

Worked example.** One heavy DNA molecule gives $2^n$ molecules after $n$ generations in ${}^{14}$N, and exactly $2$ of them are always hybrid. After $3$ generations there are $2^3=8$ molecules — $2$ hybrid and $6$ light, a ratio of $1:3$.

An everyday example. Photocopying each half of a torn page onto a new sheet gives two copies that each keep one original half.

The substance. Conservative replication would have given separate heavy and light bands after one generation — the single hybrid band ruled it out.

Replication begins at an origin of replication, where the strands separate into a replication fork; DNA-dependent DNA polymerase adds nucleotides only in the 5' to 3' direction, so the leading strand is made continuously while the lagging strand is made in short Okazaki fragments that DNA ligase joins.

The machinery:

- DNA-dependent DNA polymerase — the main enzyme; it uses deoxynucleoside triphosphates as both substrate and energy source, and works fast and accurately
- DNA ligase — seals the gaps between fragments

Why synthesis is discontinuous:

- Polymerase can build only 5' to 3'
- On the template with 3' to 5' polarity, the new strand grows continuously — the leading strand
- On the template with 5' to 3' polarity, it grows in short pieces, the Okazaki fragments — the lagging strand

Worked example. E. coli copies about $4.6\times 10^6$ base pairs in about $18$ minutes:

$$\frac{4.6\times 10^6}{18\times 60}\approx 4.3\times 10^3\text{ base pairs per second}$$

An everyday example. Painting a long wall while walking backwards — in one direction you paint smoothly, but in the other you must keep stopping and restarting in short sections, like the lagging strand.

The substance. Okazaki fragments arise from the polymerase's fixed direction, not from any defect in the lagging template.

A transcription unit has a promoter, a structural gene and a terminator; prokaryotes use a single RNA polymerase and translate their mRNA directly, while eukaryotes use three RNA polymerases and process their primary transcript, hnRNA, by splicing, capping and tailing.

Transcription unit:

- Promoter — upstream of the structural gene, where RNA polymerase binds
- Structural gene — the copied stretch; the template strand has 3' to 5' polarity, and the coding strand has the same sequence as the RNA, with T in place of U
- Terminator — downstream, marking where transcription stops

Prokaryotes:

- One DNA-dependent RNA polymerase makes all types of RNA
- mRNA needs no processing, so translation can start before transcription ends

Eukaryotes:

- RNA polymerase I — rRNAs
- RNA polymerase II — hnRNA, the precursor of mRNA
- RNA polymerase III — tRNA, 5S rRNA and snRNAs
- Splicing removes introns and joins exons
- Capping adds methyl guanosine triphosphate at the 5' end; tailing adds adenylate residues at the 3' end

Roles of RNA: mRNA carries the code, tRNA brings amino acids and reads codons, and rRNA forms ribosomes and catalyses peptide bonds.

An everyday example. Cutting the advertisements out of a recorded programme before watching it is like splicing introns out of hnRNA.

The substance. Split genes with introns are a eukaryotic feature, generally absent from prokaryotes.

The genetic code is a triplet code of 64 codons — 61 for amino acids and 3 stop signals; tRNA adapters match codons to amino acids, and translation runs through initiation at AUG, elongation and termination at a stop codon.

Features of the code:

- Triplet — three bases make one codon, giving $4^3=64$ codons
- 61 codons code for amino acids; UAA, UAG and UGA are stop codons
- Unambiguous — each codon codes for only one amino acid
- Degenerate — some amino acids have more than one codon
- Nearly universal, with a few exceptions such as in mitochondria
- AUG codes for methionine and also acts as the start codon

tRNA as adapter. Each tRNA has an anticodon loop complementary to a codon and an amino acid acceptor end.

Translation:

- Initiation — the ribosome binds mRNA at the start codon AUG, recognised by the initiator tRNA
- Elongation — charged tRNAs arrive in sequence, and the ribosome joins amino acids by peptide bonds, with rRNA acting as the catalyst
- Termination — at a stop codon, a release factor frees the finished polypeptide

An everyday example. An interpreter translating a speech word by word — tRNA matches each codon to its amino acid.

The substance. Inserting a single base shifts every later codon, which is why the code must be read in a fixed frame.