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1

Nuclear RNA Polymerases

Eukaryotes have three nuclear RNA polymerases. They are structurally similar to prokaryotic RNAPs, consisting of the same subunits. They are, however, much larger and more complex.

Eukaryotic cells have three distinct nuclear RNAPs, which each have a specific set of genes they transcribe. Each polymerase contains subunits related to those of the core enzyme in E. coli, plus a number of additional proteins.

2

Are eukaryotic RNAPs identical?

It has a large β and β' subunits and subunits similar to the α and ω subunits. However, the eukaryotic RNAPs are not all structurally identical, i.e. pol I is different from pol II.

Notably, the largest, catalytic subunit of RNA pol II (β) has a long Carboxy-terminal extension know as the C-terminal domain (CTD). It is important for various molecular interaction, allowing different complexes to interact and combine to the polymerase as it is transcribing precursor information.

3

Pol I

18S, 25S and 5.8S rRNA

Transcribes the rDNA repeat (the DNA sequence that codes for ribosomal RNA). The majority of ribosomes are made from one large transcript made by pol I.

4

Pol II

mRNA, small stable RNAs

Transcribes protein-coding genes and some small stable RNAs.

5

Pol III

5S rRNA, tRNA
Transcribes tRNA genes, 5S rRNA, a small subunit incorporated into ribosomes, and other stable RNAs

6

Do nuclear polymerases transcribe mitochondrial and chloroplast genes?

Mitochondrial and chloroplast genes are transcribed by their own polymerases. Not the same as the nuclear polymerases.

7

Transcription factors

Unlike prokaryotes, eukaryotic RNAPs do not use sigma factors, instead the recognition of the promotor region is dependent upon transcription factors.

General and specific transcription factors

8

General transcription factors

General transcription factors are required for the transcription of all pol II transcribed genes.
-They facilitate the assembly of the RNAP into a 'preinitiation complex' on the promotor.

9

Specific transcription factors

Specific transcription factors are additional transcription factors that can influence the expression of particular genes; they can stimulate and inhibit certain subsets of genes.

10

TATA Box

The promoters of many protein-coding genes contain a 'TATA box' similar in sequence to the -10 region in prokaryotes.

The general transcription factor TFIID contains the TATA-box binding protein, TBP. When it binds, the DNA bends, this is important in assembly of other proteins on the complex.

After TFIID binding, pol II then binds with TFIIF, followed by TFIIE and TFIIH.

TFIIH contains a helicase that opens up the DNA structure during initiation. It melts the DNA duplex, allowing priming.

11

Transcription Termination

Termination of transcription is required because we don't need the ribosome running along the chromosome continuously. We also need RNA to be folded into a functional form; if not it will be stable and would be degraded by the cell otherwise. In addition, lots of RNA would be created and may inhibit more of it being made.

Pol III terminates transcription at stretches of As in the template sequence, similar to intrinsic terminators in E. coli.

Torpedo model

12

Torpedo model of transcription termination

Termination of transcription by pol II involves a 'torpedo mechanism'.

Contranscriptional cleavage of the nascent RNA by an endoribonuclease releases the transcribed RNA. The cleavage has a monophosphate group and makes the ENA sensitive to nucleases, enabling the 5' to 3' exoribonuclease, Xrn2, to degrade the downstream fragment.

Xrn2 progresses along the RNA and displaces pol II from the DNA, after 'catching up' with it. The non coded tail on the RNA makes it sensitive to degradation.

13

mRNA 3' processing

Pol II termination is coupled with mRNA 3' processing. Modification of the RNA made by pol II are often required before the mRNA is functional. These are called post-transcription modifications.

5' cap structure
Splicing
Poly(A) tail

14

5' Cap structure

All poll II transcripts are 'capped' at their 5' end. Addition of the cap structure occurs shortly after transcription initiation. The cap structure is a set of nucleotides that are non-coded in a DNA sequence. They are added to the 5' end to protect the RNA from degradation.

15

Splicing

Many pol II transcripts undergo splicing, which involves remove internal 'intronic' sequences. This is additional sequences within the DNA that doesn't code for any proteins. They need to be removed from the primary transcript to generate a mRNA molecule that has a continuous sequence that can be converted into a protein.

Splicing is carried out by a large complex called a spliceosome, consisting of RNA and protein (a ribonucleoprotein complex).

16

Poly(A) tail

Non-coded adenylate residues are added to most mRNA transcripts through a two step process, involving cleavage of the transcript and polyadenylation (tail of A residues added on the end, like a tail) of the released 5' fragment.
-3' end processed of mRNA is carried out by a large complex containing cleavage factors, specificity factors and the polyadenylation machinery, known as the 'cleavage/polyadenylation complex'.
-Poly(A) polymerase is an example of a template-independent RNA polymerase, it is only able to add A residues onto RNA.

17

Transcriptional regulation

Functionally related genes are co-ordinately regulated, this may involve transcribing genes that are all needed for one process and are together in a polycistronic transcript. Expression is regulated by the encoding of only one transcript. They can be switched on or off and this allows multiple proteins to be transcribed at one. This is known as being 'transcriptionally regulated'.
However, most transcripts in eukaryotes are monocistronic, and tend to encode only a single polypeptide.

18

Transcription and translation coupling in eukaryotes?

NO
These processes are spatially compartmentalised in eukaryotes; transcription occurs in the nucleus, whereas translation occurs in the cytoplasm.

The eukaryotic mRNA is therefore generated as a distinct physical complex that must be exported to the cytoplasm for translation. This spatial separation of transcription and translation allows the transcript to undergo nuclear processing events. This allows gene expression to be regulated and manipulated. It also means cells need more time to respond to changes in their environment.

19

Transcription and RNA processing coupling

One reason the RNA splicing and 3' end processing complexes are so large is because the pre-mRNA processing is coupled to transcription. The complexes associate with the pol II enzyme in a temporarily controlled manner during the process of transcription.

The CTD (carboxyl-terminal domain on Pol II) compromises multiple copies of a serine- and threonine-rich (OH-rich) heptapeptide repeat (7 amino acid sequence; YSPTSPS). Serine residues of the CTD are phosphorylated in a way that varies during transcription of a gene. Pol II complexes that are transcribing protein-coding genes are phosphorylated at Ser-5 at the 5' end of the gene, and phosphorylated at Ser-2 at the 3' end of the gene.

The different phosphorylation patterns allow recruitment of different processing complexes at the beginning (capping complex), middle (spliceosome) and end (cleavage/polyadenylation) of transcription. This allows the processing events to occur at the appropriate time.