Transcription Process
RNA polymerase is responsible for the transcription of all RNAs. RNA polymerase fron prokaryote is a complex holoenzyme with 5 polypeptide sub units: 2 alpha, 1 beta , 1 beta’ and 1 sigma factor. The enzyme without sigma factor is referred to as core enzyme.
Transcription involves 3 different stages:
1. Initiation
2. Elongation
3. Termination
Initiation
Initiation of transcription involves the binding of RNA polymerase (holoenyme to a region of DNA. The specific region is known as promoter region. In promoter region, polymerase complex undergoes structural changes so that the DNA around the point will start unwinding and the base pairs are disrupted, producing a bubble off ssDNA. Two characteristic nucleotide sequences or base sequence in coding strand are recognized by RNA polymerase (Holoenzyme) which are:
i. Prinbow box: This consists of 6 nucleotide bases (TATAAT), located on the left side about 10 bases upstream from starting point.
ii. -35 sequences: A second sequence located about 35 bases upstream the transcription start point. Base sequences are TTGACA.
Elongation
After the promoter region has been recognized by the holoenzyjme, RNA polymerase begins to synthesize the transcript of the DNA sequence and sigma subunit is released. RNA is synthesized from 5’ to 3’ end antiparallel to DNA template. RNA polymerase utilizes ribonucleoside Triphosphate (ATP, GTP, CTP and UTP) and release pyrophosphate each time a nucleotide is added to the growing chain. The nucleotide bases in mRNA synthesized is complementary to template DNA and identical to that of coding strand.
RNA polymerase does not need prier and doesn’t possess endo or exonuclease activity. As the transcription bubble moves left to right, the DNA is unwinded ahead and rewinded behind as RNA transcribed.
Termination
Once the polymerase has transcribed the length of the gene’ it must stop and release the RNA product. This is called termination. In bacterias terminator comes in two types:
i. Rho Independent
ii. Rho dependent
Rho Independent Terminator
This is also called intrinsic terminator which consists of two sequence elements.
i. A short inverted repeats of about 20 nucleotides followed by a stretch of about eight AT base pairs plays abd important role. When a polymerase transcribe and inverted repeat sequence, the resulting RNA can form a stem loop structure (often called hairpin) by base pairing with itself. Near the base of the stem of hairpin, a sequence occur ie. Rich in G and C which is responsible for stabilization of hairpin structure. This stable hairpin slows down the progress of RNA polymerase and causes it to pause.
ii. The hairpin only work as an efficient termination when it is followed by a stretch of AU base pairs. As AU base pairs are the weaker base pairs , they are more disrupted by the effect of the stem loop on the transcribing polymerase and so the RNA will more easily be disrupted.
Rho Dependent TerminatorA specific protein rho factor binds to a C- rich region near 3’ end of growing RNA and migrates in 5’ to 3’ direction until it reaches the transcription complex that is paused at a termination site with the release of transcript RNA. The rho protein has ATP dependent RNA-DNA helicase activity.