Transcription in Pro- & eukaryotes

1.Definition
2.Transcription is selective
3.Transcription in Prokaryotes
•Initiation
•Elongation
•RNA polymerase vs DNA polymerase
•Termination
4.Transcription in Eukaryotes
•Initiation
•Elongation
•Termination
•Post transcriptional modifications

1. Transcription By Nurulhasan Khatri

2. Transcription is a process in which ribonucleic acid (RNA) is synthesized from DNA. Transcription is Selective: • The entire molecule of DNA is not expressed in transcription. • RNAs are synthesized only for some selected regions of DNA.  There exist certain differences in the transcription between prokaryotes and eukaryotes.

3. Transcription in Prokaryotes: Initiation: The binding of the enzyme RNA polymerase to DNA is the prerequisite for the transcription to start. The specific region on the DNA where the enzyme binds is known as promoter region. A single enzyme—RNA polymerase — synthesizes all the RNAs in prokaryotes. RNA polymerase of E. coli is a complex holoenzyme with five polypeptide subunits— 2 α, 1β and 1 β’ and one sigma ( σ) factor. The enzyme without sigma factor is referred to as core enzyme. There are two base sequences on the coding DNA strand which the sigma factor of RNA polymerase can recognize for initiation of transcription. 1. Pribnow box (TATA box): 6 nucleotide bases (TATAAT),10 bases upstream to the starting point of transcription. 2. The ‘-35’ sequence: base sequence TTGACA RNA polymerase of E. Coli.

4. Transcription in Prokaryotes: Elongation: As the holoenzyme, RNA polymerase recognizes the promoter region, the sigma factor is released and transcription proceeds. RNA is synthesized from 5′ end to 3′ end (5’→3′) antiparallel to the DNA template. The genetic information stored in DNA is expressed through RNA. For this purpose, one of the two strands of DNA serves as a template (non-coding strand or sense strand) and produces working copies of RNA molecules. The other DNA strand which does not participate in transcription is referred to as coding strand or antisense strand. RNA polymerase utilizes ribo-nucleotide triphosphates (ATP, GTP, CTP and UTP) for the formation of RNA. For the addition of each nucleotide to the growing chain, a pyrophosphate moiety is released. The sequence of nucleotide bases in the mRNA is complementary to the template DNA strand.

5. RNA polymerase vs DNA polymerase (RNA polymerase differs from DNA polymerase in two aspects. No primer is required for RNA polymerase and, further, this enzyme does not possess endo- or exonuclease activity. Due to lack of the latter function (proof-reading activity), RNA polymerase has no ability to repair the mistakes in the RNA synthesized. This is in contrast to DNA replication which is carried out with high fidelity. It is, however, fortunate that mistakes in RNA synthesis are less dangerous, since they are not transmitted to the daughter cells. The double helical structure of DNA unwinds as the transcription goes on, resulting in supercoils. The problem of supercoils is overcome by topoisomerases. )

6. Termination: The process of transcription stops by termination signals. Two types of termination are identified. 1. Rho ( ρ) dependent termination: • A specific protein, named ρ factor, binds to the growing RNA (and not to RNA polymerase) and in the bound state it acts as ATPase and terminates transcription and releases RNA. • The ρ factor is also responsible for the dissociation of RNA polymerase from DNA. 2. Rho( ρ) independent termination: • The termi­nation in this case is brought about by the formation of hairpins of newly synthesized RNA. This occurs due to the presence of palindromes. Transcription in Prokaryotes:

7. ρ dependent termination ρ Independent termination

8. RNA synthesis in eukaryotes is a much more complicated process than the transcription in prokaryotes. RNA Polymerases: 1. RNA polymerase I is responsible for the synthesis of precursors for the large ribosomal RNAs. 2. RNA polymerase II synthesizes the precursors for mRNAs and small nuclear RNAs. 3. RNA polymerase III participates in the formation of tRNAs and small ribosomal RNAs. Besides the three RNA polymerases found in the nucleus, there also exists a mitochondrial RNA polymerase in eukaryotes. The latter resembles prokaryotic RNA polymerase in structure and function. Promoter Sites: 1.Hogness box (or TATA box) - 25 nucleotides away upstream from the starting site of mRNA synthesis. 2. CAAT box -between 70 and 80 nucleotides upstream from the start of transcription. Etc. Transcription in Eukaryotes:

9. Initiation: RNA Pol II does not contain a subunit similar to the prokaryotic factor, which can recognize the promoter and unwind the DNA double helix. In eukaryotes, these two functions are carried out by a set of proteins called general transcription factors. The RNA Pol II is associated with six general transcription factors, designated as TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH. TFIID consists of TBP (TATA-box binding protein). The role of TBP is to bind the core promoter. The transcription factor which catalyzes DNA melting is TFIIH (Helicase) . However, before TFIIH can unwind DNA, the RNA Pol II and at least five general transcription factors have to form a pre-initiation complex (PIC). Transcription in Eukaryotes:

10. Elongation: (same as prokaryotes) The carboxyl-terminal domain (CTD) of the largest subunit of RNA Pol II is critical for elongation. During elongation it has to be phosphorylated by TFIIH with kinase activity. Transcription in Eukaryotes:

11. Termination: In the case of protein-encoding genes, the cleavage site which determines the “end” of the synthesizing mRNA occurs between an upstream AAUAAA sequence and a downstream GU-rich sequence separated by about 40-60 nucleotides in the synthesizing mRNA. Once both of these sequences have been transcribed, a protein called CPSF(Cleavage Polyadenylation Specificity Factor) in humans binds the AAUAAA sequence and a protein called CstF(Cleavage stimulating Factor) in humans binds the GU-rich sequence. These two proteins form the base of a complicated protein complex that forms in this region before CPSF cleaves the nascent pre-mRNA at a site 10-30 nucleotides downstream from the AAUAAA site. The Poly(A) Polymerase enzyme which catalyzes the addition of a 3′ poly-A tail (Polyadenylation) on the pre-mRNA. Transcription in Eukaryotes:

12. Post-transcriptional Modifications

13. Post-transcriptional Modifications This process is required to convert the RNAs into the active forms. A group of enzymes, namely ribonucleases, are responsible for the processing of tRNAs and rRNAs of both prokaryotes and eukaryotes. Messenger RNA: •The primary transcript of mRNA is the hnRNA in eukaryotes, which is subjected to many changes before functional mRNA is produced. 1. The 5′ capping: The 5′ end of mRNA is capped with 7-methylguanosine by an unusual 5’→5′ triphosphate linkage. S-Adenosylmethionine is the donor of methyl group. This cap is required for translation, besides stabilizing the structure of mRNA.

14. 2. Poly-A tail: A large number of eukaryotic mRNAs possess an adenine nucleotide chain at the 3′-end. This polyadenylation is a Template independent polymerization of mRNA . It occurs to stabilize mRNA. However, poly-A chain gets reduced as the mRNA enters cytosol. Post-transcriptional Modifications

15. 3. Introns and their removal: Introns are the intervening nucleotide sequences in mRNA which do not code for proteins. Ex ons of mRNA possess genetic code and are responsible for protein synthesis. The removal of introns is promoted by small nuclear ribonucleoprotein particles (snRNPs). snRNPs, (pronounced as snurps) in turn, are formed by the association of small nuclear RNA (snRNA) with proteins. The term spliceosome is used to represent the snRNP association with hnRNA at the exon-intron junction. Post-transcriptional modifications of mRNA occurs in the nucleus. The mature RNA then enters the cytosol to perform its function (translation). Post-transcriptional Modifications

16. Splicing

17. Thank You . By Nurulhasan Khatri