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6. Beskrive hvordan transkriptionsprocessen (bakterier og højere organismer) involverer: RNA polymerase, promotorsekvens i DNA, DNA unwinding (negativ supercoiling), DNA template strand, nukleotid triphosphat (NTP) substrater, initiering, elongering og terminering af transkription
Stryer, s. 783-789, fig. 28, 7; fig. 28, 8; fig. 28-10; fig. 28,12
Devlin, s.208-214, fig. 5.2; fig. 5.3; fig. 5.4

RNA – polymerase:

RNA polymerase performs multiple functions in the transcription 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.
    

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. 

It is important to mention that RNA polymerase has a very high processivity, meaning once it is bound to the template DNA, it won’t let go until the entire gene is transcribed.

Another characteristic of RNA polymerase is that it has no proofreading ability. This leads to higher errors rates, ca. 10⁵ times higher then DNA polymerase, but this is still acceptable since the errors are not given transmitted to progeny.

Promoter sequence in DNA - a specific sequence on the template strand of DNA that directs the RNA polymerase to the initiations site. In prokaryotes there are two promoter sequences, located 10 and 35 nucleotides upstream the start site of transcription. They have a consensus sequence, which has been depict by comparing the sequences of many prokaryotes.

Prokaryotic transcription begins with binding RNA polymerase recognising and binding its  σ -factor to a gene’s promoter sequence. The core enzyme is unable to start transcription at the promoter site.

The promoter sequence also regulates the transcription, depending on if it is a weak or strong promoter. A strong promoter has a sequence that corresponds in high degree to the consensus sequence.

In eukaryotes, there are three important consensus sequences: the TATA box, the CAAT box and the GC box. They all lie upstream from the start site, the TATA box lying closest. These promoter sequences do not bind the RNA polymerase directly as in prokaryotes; rather they bind to a transcription factor, which binds to other transcription factors and RNA polymerase II making a huge enzyme complex.

DNA-unwinding: Although RNA polymerase can search for promoter sequences when bound to double helical DNA, a region of the duplex helix must be unwinded before synthesis can begin. A region of duplex DNA must be unpaired so that nucleotides on one of its strands become accessible for base-pairing with incoming ribonucleoside triphosphates.

It is the RNA polymerase that unwinds the DNA helix. It has been estimated that each bound RNA polymerase molecule unwinds a 17-bp segment of DNA, which corresponds to 1.6 turns of B-DNA helix.

 The state in which DNA is double-helical is called a closed promoter complex, while the single stranded DNA is the open promoter complex.   

If the RNA chain would be copied without opening of the DNA double helix, the transcription complex and the newly synthesized RNA chain would have to wind around the double helix as they travel from the begging towards the end of the gene.

DNA template: A DNA template is, of course, necessary for the transcription process. The DNA strand which actually contains the gene sequence is called the coding (sense) strand. The template strand is complementary to both the coding DNA strand and the newly synthesized RNA strand, and is called the anti-coding strand.
This basically means that RNA polymerase uses the anticoding strand as a template.

This also means that the newly synthesized RNA and the coding DNA have the same sequence; just that RNA has U instead of T.

Nucleoside triphosphate substrates: All four nucleoside triphosphates: ATP, GTP, UTP, and CTP are necessary for the RNA polymerase. The principle of linking of their linking is the same as in the replication process. The 3´-OH group at the terminus of the growing RNA chain makes a nucleophilic attack on the innermost phosphate of the incoming nucleoside triphosphate with the release of a pyrophosphate – PPi.

Initiation: The initiation process requires that RNA polymerase has recognized the promoter sequence and bound its σ-factor to the promoter sequence. All this happens in a closed promoter complex, meaning the DNA helix is still intact. RNA polymerase then binds more tightly to the promoter sequence and the DNA helix is unwinded, creating the open promoter complex.

The DNA helix is unwinded in the -10 nucleotide promoter sequence, because the consensus sequence is rich in T and A, that make double H-bonds to the complementary strand, which are more easily broken.

The σ-factor is at this point still bonded to RNA polymerase. 

The unwinded DNA-strand binds the first incoming, meaning the initiating ribonucleotide and RNA polymerase then forms the first phosphodiester bond. This marks the beginning of the elongation.

Elongation: The elongation process begins with the formation of the first phosphodiester bond. After the eighth or ninth phosphodiester bond, the s factor disassociates from the RNA polymerase, which promotes a tighter bond between the RNA polymerase and the DNA template. 

As RNA polymerase continues down the double helix, it continues to separate the two strands of the DNA template. The region containing RNA polymerase, DNA template and the nascent RNA is called the transcription bubble. The newly synthesized RNA forms a hybrid complex, with the template DNA-strand. This RNA-DNA helix is about 8 bp long.

The lengths of the RNA-DNA hybrid and of the unwinded DNA region remain constant during transcription. This means that DNA is rewinded at the back at the same rate as it is unwinded in the front. It also means that the RNA-DNA hybrid is separated in the back as a new ribonucleotide is added in the front. This is all being catalysed by RNA polymerase.

The process of unwinding and restoring the DNA double helix is aided by DNA topoisomerase I and II, which are components of the transcription complex.

Topoisomerase I relaxes the positive supercoils introduced by the local unwinding of the helix, because it removes the negative supercoils introduced by the RNA polymerase on the site of transcription. This means that topoisomerase I goes in front and behind the transcription bubble.

Topoisomerase II introduces negative supercoils in DNA and can increase the efficiency of promoters on distant sites. 

During the elongation process, it is only one RNA polymerase that transcribes the entire gene, meaning RNA polymerase has a very high processivity and it won’t let go until the entire gene is transcribed.

Termniation:

The termination of the transcription can be ρ -dependant or ρ - independent.

ρ -independent termination:

The template DNA (the anticoding strand) contains stop signals. These contain a GC-rich palindromic sequence followed by an AT-rich region.

The GC-palindromic region transcribed into RNA is self-complimentary, so it makes a very stable hairpin, since the C and G form three hydrogen bonds. RNA polymerase seems to release the DNA-RNA hybrid as soon it has encountered a hairpin.

The AT-rich sequence on the DNA template is transcribed into a U-rich sequence on the RNA strand. The bonds between the RNA and DNA hybrid are now the ones between rU – dA. They are the weakest of all, which leads to disassociation of the RNA from DNA and release of the RNA polymerase.

After that, the template DNA pairs with the coding DNA and the transcription bubble closes.  

ρ - dependent termination:
Stryer, s. 789 - fig.28.12

The ρ-factor is a hexameric protein that has an essential RNA-dependant ATP-ase activity. It hydrolyses ATP only in the presence of a single stranded RNA, but not in the presence of DNA or duplex RNA. The ρ - factor is brought into action by sequences located on the nascent RNA rich in C and poor in G. 

The ρ - factor binds RNA in that way that the RNA molecule passes through the center of the protein structure.  The ATP-ase activity of the ρ - factor enables the protein to pull the nascent RNA while pursuing the RNA polymerase. When ρ catches RNA polymerase at the transcription bubble, it breaks the RNA-DNA hybrid by functioning as a helicase. 

There are 2 major differences in the transcription process in eukaryotes and prokaryotes:

• In eukaryotes, transcription takes place in the nucleus, while translation happens in the cytoplasm a little bit later. In prokaryotes, which have no nucleus, translation of the RNA begins while the transcription still goes on.

• Eukaryotes very extensively process nascent mRNA, and prokaryotes do not. Nearly all mRNA precursors in eukaryotes are spliced.

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