Question

Describe the eukaryotic RNAPII and contrast it to the prokaryotic RNAP. What additional elements are present in RNAPII, and how do these contribute to transcription?

Answer 1

Eukaryotic RNAPII:-

It is a multiprotein complex. It is an RNAP enzyme found in the nucleus of eukaryotic cells. It catalyzes the transcription of DNA to synthesize precursors of mRNA and most snRNAand microRNA. It is a 550 kDa complex of 12 subunits. RNA polymerase II is a main enzyme which catalyses DNA-directed mRNA synthesis, during the transcription of protein-coding genes.

Promoters :- In Eukaryotes promoters are the sequences which are important in the initiation of transcription of a gene . The initiation of transcription in eukaryotes requires the enzyme RNA polymerase and transcription factors, In eukaryotes the transcription factors are mainly responsible for recognizing promoters.

RNA polymeraseII promoter :-

These promoters are variable in size and are several kilobases upstream of transcription start site. The core prmoter consists of two segments , they are TATA box and Initiator or (Inr)sequences located around nucleotide (+1) . Initiators are rich in pyrimidines , some genes transcribed by RNA polymeraselI have only one of these two components of the promoter core, and some have none called null genes. Besides TATA box and Inr which act as binding site for general transcription factors, other cis acting sequences act as binding site for many regulatory factors which control expression of individual gene , they are usually found in upstream iif TATAbox.

The consensus sequence present in the core promoter (TATA box, and Inr) usually determines the location of start point, and the sequence elements found upstream influences the frequency of initiation, the elements found in any individual promoter differ in number , location and orientation . No element is common to all of the promoters.

Eukaryotic RNAPII can not initiate transcription outside a living cell, it requires many protein factors called transcription factors , it attaches to a gene regulatory site and as a result influences rate of transcription of a specific gene.

Transcription factors are of two types they are 1) Basal transcription factors 2) Regulatory transcription factors .

Basal transcription factors :- They attach to the gene promoters, they detrmine the transcription start site.

Regulatory Transcription factors - They bind to regulatory sequences like enhancers and silencers, they are either repressors or transcriptional activators.

Transcription initiation by RNAPII:- Gene transcribed by RNAPII first bind with basal or general transcription factor (GTF) , It helps in transcription from all polymerase II promoters and is part of basic transcription machinery TFIID, it is a complex made up of TATA binding protein and (TBP) and 12 TBP associated factors (TAFs). TBP is a sequence specific protein which binds to DNA by the TBP domain, which makes contact with the minor groove in the region of TATA box (TBP is universal factor, which is also required for initiation by RNApolymerase I and III) .After attachement of TFIID to the core promoter , pre initiation complex (PIC) is formed by attachement of remaining GTFs. GTFs bind to the complex in order TFIIA, TFIIB,TFIIF/RNA polymerase II ,TFIIE and TFIIH.

The final step in the assembly of initiation complex is addition of phosphate groups to the C-terminal domain (CTD) of the largest subunit of RNA PolymeraseII. CTD contains seven amino acids (Tyr-Ser-Pro-Thr-Ser-Pro-Ser) this sequence is repeated multiple times . once phosphorylated the polymerase is able to leave the pre-initiation complex and begin synthesizing RNA .

Comparision of Eukaryotic RNA polymerase with Prokaryotic RNA polymerase-

In prokaryotes whole transcription process is done by a single type of RNA polymerase.This polymerase contains four catalytic subunits and a single regulatory subunit known as sigma (s) but in eukaryotes three types of RNA POL are found RNA POL I , RNA POLII , RNA POLIII,  These polymerases differ from each other in the number and type of subunits they contain, also the type of of RNAs they transcribe, RNA pol I transcribes ribosomal RNAs (rRNAs), RNA pol II transcribes RNAs which becomes messenger RNAs (mRNAs) and also small regulatory RNAs, and RNA pol III transcribes small RNAs of transfer RNAs (tRNAs).

In eukaryotes The initiation of transcription factors requires Enzyme RNA polymerase and transcription factors , in them transcrioption factors are responsible for recognizing promoters but in prokaryotes RNA polymerase recognises the promoter.

Prokaryotic RNA polymerase can initiate transcription invitro but eukaryotic RNA polymerase can not.

The ' subunit is the largest subunit, and is encoded by the rpoC gene.it contains part of the active center responsible for RNA synthesis and contains some determinants for non-sequence-specific interactions with DNA and nascent RNA..

The Beta subunit is the second-largest subunit, and is encoded by the rpoB gene. It contains other active centers responsible for RNA synthesis and contains the rest of the determinants for non-sequence-specific interactions with DNA and nascent RNA.

The alpha subunit is the third-largest subunit and is present in two copies per molecule of RNAP, Each alpha subunit contains two domains - alpha NTD (N-Terminal domain) and alphaCTD (C-terminal domain). Alpha NTD contains determinants for assembly of RNAP., Alpha CTD (C-terminal domain) contains determinants for interaction with promoter DNA.

To bind promoters, RNAP core associates with the transcription initiation factor sigma, to form RNA polymerase holoenzyme. Sigma reduces the affinity of RNAP for nonspecific DNA and increases the specificity for promoters, allowing transcription to initiate at correct sites.

Additional elements present in RNAPII and theri contribution to transcription:-

Besides the core promoter, other cis-acting DNA sequences which regulate the RNA polymerase II transcription are, the proximal promoter, enhancers, silencers, and boundary, insulator elements . These elements contain recognition sites for many sequence-specific DNA-binding factors, which are involved in transcriptional regulation.

The proximal promoter is the region near the transcription start site . Enhancers and silencers are located many kbp away from the transcription start site and helps either to activate or to repress transcription. Boundary or insulator elements prevent the spreading of the activating effects of enhancers or the repressive effects of silencers or heterochromatin.