BIOL253 L12

What direction is RNA transcribed in?

5' to 3'. The template DNA strand is read 3' to 5'.

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The transcript has the same sequence as the...

Non template (sense) strand.

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Does transcription require a primer?

No - in comparison to replication

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What are the steps involved in transcription?

1st step involved recognition and binding of the promoter sequence by RNA polymerase. The enzyme complex separates the two strands of DNA and begins the process of synthesising the first few nucleotidees of the transcript.

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What happens one it goes past the promoter?

The complex undergoes conformational change, stabilising its interaction with DNA. The RNA polymerase continues elongating the complex, until it reaches a particular DNA sequence that reuslts in it being released from the template.

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Polymerases share common features, what are these?

A conserved core, containing 5 subunits.

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What is the core enzyme?

Everything (all subunits) except sigma factor.

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What is a holoenzmye?

It is the core enzyme and the sigma subunit (factor). The sigma factor directly contacts the promoter sequence. This contact ensures that transcription begins at the proper position in DNA.

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What does the sigma factor do?

It locates the transcriptional start site by recognising the promoter. It does this by binding to specific sequences upstream of the start site of transcription whose position is noted as +1.

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What are the key elements that are recognised by a sigma factor ?

a conserved sequence most commonly centred at 35 nucleotides upstream of the start of transcription, commonly called the minus 35 element, and a a second conserved sequence centred at 10 nuceotides upstream of the start site (minus 10) element.

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What are the -35 and - 10 sequence known as?

Consensus sequences (TTGACA AND TATAAT)

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How does sigma factor interact with DNA

Sigma factor interacts with DNA via several domains (3).

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Genes that are transcribed frequently have a promoter sequence that is...

Similar to the consensus.

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Genes that are transcribed infrequently have a promoter sequence which is...

Different to the consensus.

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What are the different types of Eukaryotic RNA polymerases?

RNA polymerase I, II and III

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Where is Polymerase I found and what does it transcribe?

Found in the nuceolous, transcribes rRNA.

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Where is polymerase II found and what does it transcribe?

Nucleoplasm: mRNA (messenger RNA for protein coding), snRNAs (small nuclear RNAs found in spliceosomes), miRNAs. (which regulate gene expression)

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Where is polymerase III found and what does it transcribe?

It is foundin the nucleoplasm. It transcribes 5S rRNA (component of large sibosomal subunit) tRNAs (small RNA adaptor molecules - place amino acid in graowing polypeptide chain) and also snRNAs (small nuclear RNAs found in spliceosomes).

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What are transcription factors?

RNA polymerase core enzymes in eukaryotes and archaea depend on additinoal proteins to target them to specific sites in DNA where transcription is initiated.

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How are the eukaryote and archaeal transcription factors different to bacterial sigma factors?

While bacterial sigma factors target the polymerase to the promoter and mediate promoter melting, the eukkaryotic and archael generla transcription factors also catalyze additional steps in the initiation of transcription.

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What are the general transcription factors associated with RNA polymerase II

TFIIA (transcription factor for RNA polymerase II, species A), TFIIB, TFIID, TFIIE, TFIIF and TFIIH.

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What does TFIIA do?

It assembles at promoters together with the 12 unit polymerase core enzyme to form the pre-initiation complex.

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The eukaryotic RNA polymerase I and III enzymes require distinct sets of proteins to form a transcription initiation complex. Just one protein is required for transcription initiation by all eukaryotic polymerases, what is it?

TATA binding protein (TBP), which is a subunit of the TFIID complex. TBP is conserved in archaea.

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The promoters of genes transcribed by RNA polymerase II commonly contain a...

TATA box (named for it's T/A,A.T/A concensus sequence, to which the TBP protein binds.

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Where is the TATA Box located?

25-30 bp to the upstream side of the transcription site.

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What is the binding site for the TFIIB?

BRE (TFIIB recognition element)

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What promotes binding of TFIIB to BRE?

Binding of TBP to TATA box.

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What happens to the remaining components?

They bind to form the pre-initiation complex

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What is one feature common among all three RNA polymerase enzymes?

Their dependence upon TBP to initiate transcription.

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How is transcription initiated?

RNA polymerases open up about 14bp of duplex DNA (Transcription bubble). In Pol II, the transcription bubble is opened by helicase subunits og TFIIH and, uniquely, requires ATP.

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What do RNA polymerases do first?

They open up about 14bp of duplex DNA (Transcription bubble).

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In pol II the transcription bubble is opened by

Helicase subunits of TFIIH and requires ATP

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What is abortive initiation?

When RNA polymerase fails to make a full length RNA on the first attemp. It leads to the release of short RNAs of 2-9 nucleotides

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How does RNA polymerase hold the DNA strands apart?

The rudder, lid and zipper help keep the two strands of DNA apart

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What is involved in abortive initiation?

Bacterial sigma factor and eukaryotic TFIIB are involved in abortibe initiation. Each has a loop the extends into the active site region. The loop is in a position to block the elongating transcript.

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Why does the loop need to be moved in order for transcription to continue?

Because the loop is in a position to block the eleongating transcript

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Displacement of the protein loop is thought to help the ...

Polymerase break away from the promoter - promoter clearance.

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What happens to the RNA polymerase when the protein loop displaces?

It undergoes a conformational change that associatess it very stable with DNA, and loosens its grip on initiation factors

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What does RNA pol II make?

It makes mRNA but this is not in the final active form, so it called the pre-mRNA

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Phosphorylation of the RPB1 CTD couples pre-mRNA processing with transcription elongation by RNA polymerase II.

The PolII is phosphorylated as it converts to the elongating complex. Phosphorylation of the 5th serine in the heptad repeat on the CTD region of the Rpb1 subunit occurs 1st. This recruits RNA processing enzymes, such as those that cap the 5' end MRN

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What happens to elongation during capping?

It is briefly paused to allow capping to occur.

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What does capping lead to?

It leads to phosphorylatin of the second serine in the CTD heptad repeat, which causes the polymerase to resume elongation.

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What results from transcription elongation?

Supercoiling . as the transcription bubble continues along the DNA positive supercoiling increases ahead of the polymerase and negative super coiling increases behind the polymerase.

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Changes in supercoiling could cause...

Stalling of RNA polymerase, and the tension must be relieved by topoisomerases.

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In E.coli, what removes the positive supercoils and what removes the negative super coils?

DNA gyrase removes the positive supercoils and DNA topoisomerase I removes the negative supercoils.

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Why is DNA hindered by chromatin?

As transcription proceeds, the DNA flaking the transcribed region is not free to rotate even in linear chromosomes due to the packaging of DNA in chromatin (it needs to be able to rotate to release the supercoils)

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What do Eukaryotes use to remove the nucleosomes ahead of RNA polymerase?

hISTONE CHAPERONES

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What do histone chaperones include?

FACT (Failitates chromosome transcription) Asf1 and Spt6.

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How does RNA polymerase correct mistakes?

Sometimes RNA polymerase stalls. It then reverses direction and the mode recently made RNA protrudes from the complx. Transcript cleavage factors chop this off through stimulating RNA polymerase endonuclease activity. This is used to correct mismatch

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What are the two types of terminators in E.coli?

Type 1 terminators aka Rho- independent or Intrinsic terminators. Type II terminators aka Rho dependent terminators.

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Do type I terminators require anything for termination?

No additional factors required for termination

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Do type II terminators require anything for termination?

ATP

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In the termination process by RNA PolII, most eukaryotic mRNAs have a...

Poly-A tail added through polyadenylation.

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What happens during Termination by RNA PolII (Allosteric model)

RNA PolII continues to transcribe after the polyadenylation signal, but the mRNA is cleaved at the polyA signal and the 3' end of the mRNA is processed.

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What is the torpedo model of RNA PolII termnination?

After cleavage, the RNA downstream of the poly A sie is digested by a 5' to 3' endonuclease (Rat1).

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Why is this model called torpedo?

The endonuclease is the torpedo. It continues to degrade the nascent RNA until it runs into the RNA polymerase (Like a torpedo). This disrupts polymerization and cuases the enzyme to dissociate from the DNA.

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BIOL253 L12

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