(34)
4. Beskrive (kortfattet) funktionen af en RNA polymerase under transkriptionen
Devlin, s.208-210
Stryer, s.130-131
Stryer, s.782

Transcription or RNA-synthesis is the process by which RNA chains are made of DNA template. DNA nucleotide sequence information is transcribed into RNA sequence information. The enzymes that catalyse the transcription process are called RNA polymerases.

The transcription process takes place in three stages: initiation, elongation and termination. RNA polymerase performs multiple functions in this process:

1. It searches DNA for initiation sites, also called promoter sites (promoters). These DNA-sequences are on the same strand as the gene itself.
    

2. It unwinds a short stretch of double-helical DNA to produce a single-stranded DNA template from which it takes instructions.
    

3. It selects the correct ribonucleoside triphosphate and catalyses the formation of a phosphodiester bond. The 3´-OH group at the terminus of the growing chain makes a nucleophilic attack on the innermost α - phosphate of the incoming nucleoside triphosphate with the release of a pyrophosphate.
    

4. It detects termination signals that specify where a transcript ends.
    

5. It interacts with transcription factors - activator and repressor proteins that modulate the rate of transcription initiation by binding to specific sequences in the DNA. 

RNA polymerase requires the following components for transcription:

1. A template - the preferred template is a double stranded DNA molecule.

2. Activated precursors – all four ribonucleosides are required: ATP, GTP, UTP and CTP.

3. A divalent metal jon, fx. Mn²⁺

The synthesis of RNA is like that of DNA in several aspects:

• The direction of the synthesis is 5´-3´on the new synthesized RNA-strand.

• The mechanism of elongation is similar: the 3´-OH group at the terminus of the growing chain makes a nucleophilic attack on the innermost phosphate of the incoming nucleoside triphosphate.

There are though two major differences in contrast with DNA polymerase:

• RNA polymerase lacks the activity of proofreading and excising mismatched nucleotides.

• RNA polymerase doesn’t require a primer in order to start the transcription.

All cellular RNA is made by RNA polymerase. The chemistry of RNA-synthesis is identical for all RNAs, but the location of synthesis and the RNA polymerase type are different.
 

Angive at transkription i prokaryote celler kun involverer en RNA polymerase, mens tre forskellige RNA polymeraser er nødvendige for eukaryot transkription
Stryer, s. 783
Stryer, s.793, table 28.2
Devlin, s.210

There is a difference between the transcription processes in prokaryotes and eukaryotes when it comes to the number of RNA polymerases involved.  

In prokaryotic cells there is only one RNA polymerase that catalyses the transcription process. I will take the RNA polymerase in E.Coli as an example.

RNA polymerase from E.Coli is a very large (ca. 400 kDa) and complex enzyme consisting of 5 subunits (a₂, b, b`, s). The enzyme can be divided in the following manner:

• The σ - subunit - that helps find the promoter site which directs the RNA polymerase to the initiation site. A large fragment of the σ-factor is an α-helix that recognises the -10 sequence (the TATAAT sequence) on the DNA template. Thus, it participates in the initiation of RNA synthesis and then disassociates from the rest of the enzyme, when the newly synthesized RNA strand has reached 9 or 10 nucleotides in length. The σ subunit plays a key role in determining where RNA polymerase initiates transcription. 
  The σ-factor is also involved in transcription regulation; if it doesn’t bind, transcription doesn’t occur.
  The σ-factor decreases RNAs affinity for random DNA for 10^-4 times. It walks on the DNA until it finds the compatible seqeuence.    

• The core enzyme – the two α subunits, the β and β` subunits that contain the catalytic site. The catalytic site resembles the DNA polymerase. The core enzyme can not start the transcription on the promoter by itself. After the s factor is lost, the core enzyme makes a tighter bond to the DNA template.  

The core enzyme and the σ - subunit form the holoenzyme, which is capable of specific RNA synthesis in vivo and in vitro.       

 In eukaryotic cells, there are three different RNA-polymerases involved.

• RNA polymerase I – located in the nucleolus, where it transcribes the tandem array of genes for 18S, 5.8S and 28 S rRNA.
   

• RNA polymerase II – located in the nucleoplasm, synthesizes the precursors of mRNA as well as several small RNA molecules, such as those of the splicing apparatus.
   

• RNA polymerase III – located also in the nucleoplasm, where it synthesizes all tRNAs and the 5.S rRNA.  

tilbage til molekylær biologi