VPB 321: Animal Biotechnology (2+1)

TRANSCRIPTION

• Transcription generates a single stranded RNA identical in sequence with one of the strands of the duplex DNA.
• Several different types of RNA are generated by transcription: the three principal classes involved in the synthesis of proteins are
  • Messenger RNA (mRNA)
  • Transfer RNA (tRNA)
  • Ribosomal RNA (rRNA)
• Transcription is the synthesis of a single stranded RNA from a double stranded DNA template. RNA synthesis occurs in the 5' 3' direction and its sequence corresponds to that of the DNA strand which is known as the sense strand. The strand of DNA that directs the synthesis of mRNA via complementary base pairing is called the template strand or antisense strand. The other strand bears the same sequence as the mRNA is called the coding strand or sense strand. View image...
• There are 3 steps involved in transcription.
• Initiation
  • RNA polymerase is the enzyme responsible for transcription. It binds to specific DNA sequences called promoters to initiate RNA synthesis. These sequences are upstream (to the 5'-end) of the region that codes for protein, and they contain short, conserved DNA sequences which are common to different promoters.
  • The RNA polymerase bind to the ds DNA at a promoter sequence, resulting in local DNA unwinding.
  • The position of the first synthesiszed base of the RNA is called the start site and is designated as position +1.
•  Elongation
  • RNA polymerase moves along the DNA and sequentially synthesizes the RNA chain.
  • DNA is unwound ahead of the moving polymerase, and the helix is reformed behind it.
• Termination
  • RNA polymerase recognises the terminator which causes no further ribonucleotides to be incorporated. This sequence is commonly a hairpin structure. Some terminators require an accessory factor called rho for termination.

E.coli RNA Polymerase

• RNA Polymerase is responsible for RNA synthesis (Transcription). A single type of RNA Polymerase is responsible for all synthesis of mRNA, rRNA and tRNA.
• The core enzyme consisting of 2 a, 1 b, 1 b ' and 1 e subunits, is responsible for transcription elongation. The sigma factor (s) is also required for correct transcription initiation.
• The complete enzyme, consisting of the core enzyme plus the s factor, is called the holoenzyme. Two alpha (a) subunits and one beta (b)subunit present in the RNA polymerase are involved in promoter binding.
• One beta prime (b')subunit present in the RNA polymerase is involved in template DNA binding.
• Sigma (s) factor is a separate component from the core enzyme and is required for initiation at the correct promoter site. View image...

The E.coli promoter

• Promoters contain conserved sequences which are required for specific binding of RNA polymerase and transcription initiation. The promoter region extends for around 40 bp.
• The -10 sequence is a 6 bp region present in almost all promoters. This hexamer is generally 10bp upstream from the start site. The consensus -10 sequence is TATAAT and is important for unwinding. This is referred to as Pribnow box.
• The -35 sequence is a further 6 bp region recognizable in most promoters. This hexamer is typically 35 bp upstream from the start site. The consensus -35 sequence is TTGACA.