TRANSCRIPTION IN PROKARYOTES AND EUKARYOTES

Transcription is the key process in the Central Dogma of Molecular biology which forms the base of genetic information flow. The synthesis of messenger RNA in both eukaryotes and prokaryotes takes place with help of this process. But due to different cell structure of eukaryotes (having nucleus) and prokaryotes (without nucleus), the process is quite different in some aspects. Without this, the genetic information flow might not get completely translated into proteins. To study the whole Transcription process, I have made this presentation as part of my class seminar. I have taken help from various resources and compiled it in an easy and understandable way. Hope you find it helpful.

1. Seminar on MECHANISM OF TRANSCRIPTION SUBMITTED BY – Anjali Thakur (M.Sc Zoology)

2. INTRODUCTION TO CENTRAL DOGMA According to this, the genetic information flows from DNA to RNA (mRNA) and from RNA to proteins. This path for the flow of genetic information has been termed by Crick .

3. CENTRAL DOGMA REVERSE The synthesis of DNA on RNA template in RNA tumour viruses was demonstrated by two American Scientists, H.Temin and D.Baltimore. This discovery introduced the concept of Central Dogma Reverse, according to which the genetic information does not necessarily flow from DNA to RNA but also from RNA to DNA.

4. TRANSCRIPTION (synthesis of mRNA) The process of formation of mRNA over the DNA template strand is called Transcription. It occurs during the Interphase. The enzyme responsible for transcription is DNA dependent RNA Polymerase which adds complementary ribonucleotides at the 3’ end of growing mRNA molecule. The synthesis only occurs in direction 5’ to 3’ direction. Along with RNA polymerase, there are some transcription factors (TF) which also plays some important roles.

5. TRANSCRIPTION IN PROKARYOTES Occurs in Cytoplasm as they lack membrane bound nucleus to enclose their genetic material. In prokaryotes, single RNA polymerase enzymes undertakes the formation of all RNAs (mRNA, tRNA, rRNA). RNA polymerase in E. coli is a large and complex enzyme made up of 5 subunits or polypeptide chains forming the Core Enzyme. Sigma factor is weakly attached with the core enzymes and forms a Holoenzyme. It plays an important role in the initiation of Transcription by identifying and binding to specific DNA sequence on the promotor , which are the start sites of the genes.

6. Omega subunit, though the smallest, plays a structural role in maintaining the proper confirmation of the largest beta’ subunit and facilitates it’s incorporation into core enzymes complex.

7. PROCESS OF TRANSCRIPTION The transcription process involves 3 stages:- INITIATION This phase of Transcription begins with recognition of specific DNA sequence called PROMOTERS having common sequence TATAATG or close derivatives of it called as PRIBNOW BOX or TATA box. At this site, the enzyme is composed of core enzymes and sigma factor. Sigma factor helps to identify and bind to specific sequence. RNA polymerase after binding to the promoter region of DNA initiates transcription process. This binding cause separation of DNA strands to expose bases for base pairing.

8. 2. ELONGATION  After the transcription process is initiated, the sigma factor gets dissociated from the core enzymes and can be reused. Elongation of RNA chains takes place by only core enzymes which move along with the DNA template. The various ribonucleosides triphosphates change them to ribonucleosides monophosphate and set free pyrophosphate group which contains high energy used for polymerization. It requires enzyme pyrophosphatase . RNA chain growth take place in the 5’-3’ direction.

9. 3. TERMINATION In prokaryotes, two primary termination pathways are well- characterized: * Rho- Dependent Termination This mechanism relies on a protein called Rho factor , an ATP-dependent helicase . It’s helicase activity unwinds the RNA-DNA hybrid leading to the dissociation of RNA polymerase from DNA template and the release of the newly synthesized RNA. It binds to specific “rut” (Rho utilization) sites on growing RNA. It uses ATP to translocate along the RNA in a 5’- 3’ direction, “chasing” the moving RNA polymerase.

10. * Rho- Independent (Intrinsic) Termination This pathway does not require any accessory protein. It is characterized by specific sequence within DNA template that lead to the formation of structures in nascent RNA molecule. These sequences typically consist of: INVERTED REPEAT SEQUENCE : or palindromic sequence. This region in newly synthesized RNA folds back on itself to form a stable hairpin loop like structure due to H-bond between GC base pairs. POLY- U TAIL : Following hairpin structure, there is a stretch of adenine (A) bases in DNA template which is transcribed into a run of Uracil (U) bases in RNA.

11. Formation of the stable hairpin loop causes the RNA polymerase to pause it’s movement along the DNA template. The weak hydrogen bonds between the poly – U tail in RNA and poly- A stretch in DNA template are not strong enough to hold the RNA- DNA hybrid together during this pause. This leads to spontaneous dissociation of the RNA transcript and RNA polymerase from the DNA template and the termination of Transcription. ** Newly formed RNA is known as Transcript mRNA because it had to be processed before sending it to the cytoplasm.

12. TRANSCRIPTION IN EUKARYOTES Occurs in nucleus. The nuclear membrane introduces a barrier between transcription and translation and therefore it must be exported to the cytoplasm before reaching the ribosomes. In eukaryotes, three distinct nuclear RNA polymerases transcribes different classes of genes. RNA polymerase I forms precursor rRNAs RNA polymerase II forms precursor mRNAs RNA polymerase III forms precursor tRNAs These mRNAs are generally monocistronic ensuring that one mRNA molecule translate into 1 specific protein.

13. Transcription in eukaryotes require RNA polymerase II enzyme along with some Transcription Factors (TF) . INITIATION:- This phase starts with recognizing specific DNA sequences in the PROMOTER sites. This site consists of DNA sequence almost identical to PRIBNOW box of prokaryotes, it is called as Hogness- Goldberg box or TATA box located about 20-30 nucleotides away (upstream) from initiation site. There also exist another site of recognition between 70-80 nucleotides upstream called as CAAT box.

14. **While prokaryotic RNA polymerase directly bind with the DNA, eukaryotic RNA polymerase II recognizes the proteins which are bind to the DNA. **The initiation process in eukaryotes starts with the formation of Pre-Initiation Complex .

15. First, the TFIID binds to the TATA element in the promoter region with help of it’s TBP protein (Tata Binding Protein). After binding, it bends the DNA by 80° which helps in binding with the other Transcription Factors which are:  TFII A - helps in stabilizing the binding of TFIID with promotor.  TFII B - binds to TBP and helps to position RNA polymerase II correctly at promotor site.  TFII F - helps in recruitment of RNA polymerase II.  TFII E - binds to the pre initiation complex.

16. TFII H-large component with 9 sub-units out of which:- 2 subunit performs the Helicase Activity which helps in unwinding of DNA strands with using energy from ATP. This activity helps in transition from Pre-initiation complex to open complex. 7 subunit performs the Kinase Activity which phosphorylates C-Terminal domain of polymerase. This activity is crucial for releasing the polymerase from promotor for proceeding into elongation phase.

17. ELONGATION:- Transcription Factors helping in elongation are called as Elongation Factors and these are:-  TFEb – Kinase protein which stimulates elongation

18. TFII S - helps in increasing the rate of transcription in areas where it is slow. These elongation factors helps in preventing the transcription process from halting permanently and allowing for the completion of mRNA synthesis. These factors helps in assisting the polymerase by helping it move forward and clear obstacles like nucleosomes.

19. TERMINATION Unlike prokaryotes, eukaryotic RNA Polymerase II transcription does not terminate at a distinct stop signal. Instead, termination is closely coupled with pre-mRNA processing, specifically the addition of a poly-A tail. KEY STEPS:- Poly (A) signal recognition:- The primary signal for this process is Poly A sequence (PAS) which signals the RNA polymerase II that Transcription is about to end. Protein Factors:- C-Terminal domain of RNA polymerase II consists of 2 proteins- CPSF: Cleavage and Polyadenylation Specificity Factor CstF: Cleavage Stimulating Factor

20. 3. Cleavage: Once, the Poly A sequence is transcribes, these factors move to the transcribed sequence on mRNA. Additional cleavage factors- endonucleases are recruited by Polymerase enzyme. Along with the help of CstF, these endonucleases help in cleavage of mRNA and gets dissociated just after cleavage. 4. Polyadenylation: The CPSF then recruits Poly A polymerase which adds 200 Adenine sequence at 3’ end of mRNA giving rise to Poly A tail. After which, Poly A Binding Protein (PBP) binds to Poly A tail and prevents degradation.

21. 5. Polymerase Dissociation (Torpedo Model) Continued Transcription – RNA polymerase II continue to transcribe a small and uncapped strand of mRNA from cleavage site. Exoribonuclease Degradation – in case of human, Xrn2 , an exoribonuclease degrades RNA from 5’ – 3’ end. Dissociation Signal- This degradation process acts like a “Torpedo” i.e. catching up to the polymerase and causing it to dissociated from DNA template. Thereby finally terminating Transcription process.

22. Heterogeneous nuclear RNA (hnRNA) Primary mRNA transcript produced by RNA polymerase II in eukaryotes is often referred to as heterogeneous nuclear RNA as they are interrupted by non- coding DNA segments. Therefore, it has to undergo some modifications needed for protein synthesis. Post – Transcriptional Modifications THE 5’ CAPPING :- first RNA processing event Terminal gamma phosphate of 5’ end nucleotide is removed by RNA triphosphatase . Guanylyl transferase enzyme carries out the reaction between beta- phosphate of 1 st nucleotide and alpha- phosphate of GTP. Once guanine is attached, methyl transferase enzyme attaches a methyl group to guanine nucleotide. This structure is called 5’ cap.

23. 2. 3’ POLYADENYLATION :-

The 3’ end of the pre-mRNA is modified by the addition of a poly(A) tail, a stretch of 50-250 adenine nucleotides. This process involves the recognition of a polyadenylation signal sequence (e.g., AAUAAA) in the pre-mRNA, followed by cleavage downstream of this sequence and the enzymatic addition of adenines by poly(A) polymerase. The poly(A) tail enhances mRNA stability, influences nuclear export, and promotes translation efficiency.

24. 3. SPLICING :- Eukaryotic genes often contain non-coding sequences called introns interspersed within coding sequences called exons . Splicing is the process by which introns are precisely removed from the pre-mRNA, and the remaining exons are ligated (joined) together.  Spliceosome Assembly :
The process is mediated by the spliceosome, a large ribonucleoprotein complex composed of proteins and snRNAs forming snRNPs , assemble at these sites.

25.  Recognition of Splice Sites :
The spliceosome recognizes specific sequences called splice sites at the 5’ and 3’ ends of an intron, as well as a branch point (an adenine nucleotide) within the intron.  Intron Removal :
The spliceosome cuts the pre-mRNA at the splice sites, causing the intron to loop out and form a lariat-like structure.  Exon Ligation :
The two exons on either side of the excised intron are then joined together with help of Ligase enzyme .

26. WHY NEED THESE MODIFICATIONS????  Produce functional RNA molecules :
The primary RNA transcript from transcription is often a precursor and must be modified to become mature and functional.  Protect RNA from degradation :
The 5’ cap and 3’ poly-A tail protect the mRNA molecule from being broken down by cellular enzymes before it reaches the ribosome.  Enable protein synthesis :
Modifications are necessary for the mRNA to be correctly recognized by the ribosome, the machinery responsible for translating the genetic code into a protein.  Remove non-coding sequences :
Introns, which are non-coding sequences, are removed from the precursor mRNA during splicing so that only the essential coding information remains.

27. DIFFERENCE BETWEEN PROKARYOTIC AND EUKARYOTIC TRANSCRIPTION PROKARYOTES It occurs in Cytoplasm. Transcription and translation are coupled. Single type of RNA polymerase synthesize all three RNAs. Post- Transcriptional Modifications do not occur. Produced mRNA are polycistronic. EUKARYOTES It occurs in nucleus. Transcription and translation occurs separately. RNA polymerase I, II and synthesize rRNA, mRNA and tRNA resp. Post- Transcriptional Modifications are necessary. Produced mRNA are monocistronic. LOCATION COUPLING RNA poly. MODIFICATION PRODUCED mRNA