Crick therefore regarded the code as a "frozen accident" - that is, incapable of further evolution [J. Mol. Biol., 1968, 38, 367].

• Many changes in the code alter the meaning of a codon, which would introduce an error into every translated message

• The probability of this error being beneficial is very small

• However, changes in the "wobble" base are often innocuous

Nonetheless, the observation that mitochondrial and and a few bacterial primary genome codes differ means that mutation is possible.

• The most likely mechanism is a mutation in the tRNA (see below)

• The most likely reassignment is of a stop codon, because they occur only once per gene

Each time a cell divides, its DNA must be replicated. Some questions to be answered:

• What is the start signal?

• What molecular machinery is required?

• How is that machinery assembled in the right place at the right time?

• How is the DNA unwound and released from the nucleosomes?

• How is the new DNA supplied with nucleosomes?

• How are the needed nucleotides delivered to the assembly line?

• What is the molecular mechanism of attachment of nucleotides to the growing polymer?

• In what direction does replication proceed?

• How is replication terminated?

Very similar questions can be asked about the transciption of DNA into RNA, and then about the translation into protein.

Here is a sketchy outline of the first step: replication of DNA

Replication, transcription, and translation are all catalyzed by enzymes. In E. coli, some 30 proteins are involved in replication, forming a structure called a replisome.

• The two parent strands are unwound by enzymes called DNA helicases
  • Supercoiling is relieved by topoisomerases

• Binding proteins attach to the single strands to prevent them from rewinding

• The point where all the action is going on is called the replication fork

• Replication proceeds with each new single strand built in the 5' ® 3' direction
  • On the strand with the free 3' end, called the leading strand, replication proceeds continuously

  • On the strand with the free 5' end, the lagging strand, replication proceeds by forming fragments, called Okazaki fragments, that must be joined later

  • An RNA primer is needed on this strand, and is build by an enzyme called a primase; it is later clipped off when the segments are joined

• The major work is done by DNA polymerases, which are complexes of multiple protein subunits; E. coli has at least four
  • Polymerases can only add nucleotides to an existing strand

  • Enzymes called primases provide a primer, usually made of RNA

Cells contain multiple helicases, which are needed for other tasks, such as DNA repair, as well as for replication and transcription.

Rep helicase is one of those found in E. coli:

Rep Helicase (1uaa) Bound to polyT

• In solution, Rep is a monomer; two copies bind to DNA (blue and orange)

• The two copies alternately bind to the double strand, separating it, and to the single strand

• A substantial conformational change accompanies binding to the single strand

Superposition of Open (blue) and Closed (orange) Rep

Like most helicases, Rep "burns" adenosine triphosphate (ATP) to provide the energy for unwinding DNA.

The single stranded DNA produced by the helicases must be kept from:

• Rewinding to double strand DNA

• Self-pairing, like RNA

Single strand binding proteins (SSB) maintain the single strands.

SSB (1eyg) from E. coli Bound to PolyC

• The protein is a homotetramer

• Most commonly, only two subunits bind DNA

• This mode forms strings of contacting SSBs along the length of unwound DNA