Prokaryotic Transcription

Transcription in Bacteria (Prokaryotes)

• The site at which transcription should start is designated "+1."
• RNA Polymerase has a subunit called Sigma factor that can bind to specific DNA sequences within the promoter region of a gene.

Initiation

1. The sigma subunit binds to the -35 box (~35 bases upstream from the +1 site, where transcription begins) and the -10 box (~10 bases upstream from the +1 site) in the promoter region.
2. The remaining subunits of RNA polymerase can then bind to the promoter. The RNA polymerase complex is referred to as a holoenzyme.
3. The RNA polymerase is able to orient itself near the +1 site and to move in the direction of transcription.
4. Binds to template strand.
5. It reads DNA 3' to 5'.
6. It synthesizes RNA in 5' to 3'.

• The promoter sequence, including the -35 box and -10 box, can vary between genes and bacteria, but are still recognized by Sigma because they are relatively similar.
• Comparing the sequences for the -35 box and -10 box of different genes can yield a consensus sequence, or the most common nucleotide at a particular position.
• The -35 region has the consensus sequence TTGACG and the -10 region has the sequence TATAAT.
• The closer to consensus a sequence is, the higher the affinity the Sigma has for the promoter.

Elongation

• Elongation can start when the polymerase releases the sigma subunit can move forward.
• The RNA polymerase, orientated at the +1 site, moves along the template strand in the 3' to 5' direction and creates a complementary RNA copy by base pairing (because RNA nucleotides are used, uracil replaces thymine).
• The RNA strand is created complementary to the template strand and is identical to the coding (non- template) strand.

Termination

1. Rho-dependent termination: uses a protein called Rho that binds to the new RNA strand and causes RNA Polymerase to fall off upon their encounter.
2. Formation of a hairpin (Rho-independent):
3. The RNA polymerase reaches a termination sequence in the DNA; this is typically a region with lots of C and G nucleotides and a poly A sequence.
4. The region with C-G folds onto itself to form a hairpin loop. This causes polymerase to pause.
5. The region with a run of A-U is not very stable, and in combination with the hairpin, causes the polymerase to dissociate.