Semiconservative DNA Replication

The Watson-Crick model immediately suggested how DNA copies itself: the two strands separate and each acts as a template for making a new complementary strand. Each daughter molecule thus has one old (parental) strand and one new strand - so replication is semiconservative.

The Meselson-Stahl experiment (1958)

Matthew Meselson and Franklin Stahl proved this in E. coli:

  • They grew E. coli for many generations in a medium with heavy nitrogen (15N) as the only nitrogen source, so the DNA became uniformly heavy.
  • The cells were then shifted to a medium with normal light nitrogen (14N), and DNA was extracted after each generation and spun in a caesium chloride (CsCl) density gradient.
  • After one generation the DNA was all of intermediate (hybrid) density (one 15N strand + one 14N strand); after two generations, half was hybrid and half was light. This is exactly what semiconservative replication predicts.

Meselson-Stahl experiment showing semiconservative DNA replication using heavy and light nitrogen

A similar result was obtained by Taylor and colleagues on chromosomes of the broad bean, Vicia faba, using radioactive thymidine.

One-liners: replication is semiconservative (each daughter = 1 old + 1 new strand); Meselson & Stahl used 15N/14N + CsCl density gradient in E. coli; Taylor showed it in Vicia faba.

The Replication Machinery

In eukaryotes, replication happens in the S phase of the cell cycle. The main enzyme is the DNA-dependent DNA polymerase, which is remarkably fast and accurate.

  • Replication begins at a specific site, the origin of replication (ori), where the helix opens to form a replication fork.
  • DNA polymerase can add nucleotides only in the 5' to 3' direction, reading the template 3' to 5'.
  • Because of this one-way rule, the two template strands are copied differently:
  • the strand whose template runs 3' to 5' towards the fork is made continuously - the leading strand;
  • the other is made discontinuously as short Okazaki fragments - the lagging strand - which are later joined by DNA ligase.
  • Replication also needs an RNA primer to start, and the deoxyribonucleoside triphosphates (dNTPs) act both as the substrates and as the source of energy for polymerisation.

Replication fork showing leading strand, lagging strand, Okazaki fragments, DNA polymerase and ligase

One-liners: main enzyme = DNA-dependent DNA polymerase (adds only 5' to 3'); leading strand continuous, lagging strand = Okazaki fragments joined by DNA ligase; needs a primer; dNTPs = substrate + energy; replication in the S phase.

Transcription

Transcription is the copying of the information in one strand of DNA into RNA. Only a segment of DNA and only one of the two strands is copied - if both strands were copied, they would code for different proteins, and the two complementary RNAs would pair and not be translated.

  • The strand that is copied (read 3' to 5') is the template strand; the other strand, which has the same sequence as the RNA (with T in place of U) and runs 5' to 3', is the coding strand.
  • A transcription unit has three parts: a promoter (where RNA polymerase binds, upstream of the gene), the structural gene, and a terminator (downstream).

RNA polymerases

  • In bacteria, a single RNA polymerase makes all types of RNA; the sigma factor helps initiation and the rho factor helps termination.
  • In eukaryotes there are three RNA polymerases: RNA polymerase I makes rRNAs; RNA polymerase II makes the mRNA precursor (hnRNA); RNA polymerase III makes tRNA, 5S rRNA and small nuclear RNAs.

RNA processing in eukaryotes

In eukaryotes the primary transcript (hnRNA) is non-functional and must be processed in the nucleus:

  • Splicing - the non-coding introns are removed and the coding exons are joined.
  • Capping - a methyl-guanosine cap is added at the 5' end.
  • Tailing - a poly-A tail is added at the 3' end.

The mature mRNA is then transported out. (Bacterial mRNA is often polycistronic; eukaryotic mRNA is monocistronic, and the genes are split into exons and introns.)

One-liners: only one strand (template, 3'->5') copied; coding strand = same as RNA (U->T); transcription unit = promoter + structural gene + terminator; bacteria one RNA pol (sigma = initiation, rho = termination); eukaryotes RNA pol I/II/III (rRNA / hnRNA-mRNA / tRNA); processing = splicing + capping + tailing.