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Similar to transcription in prokaryotes and eukaryotes (20)
transcription in prokaryotes and eukaryotes
- 1. TRANSCRIPTION • Transcription is the synthesis of RNA chain from one of the strands of duplex DNA. • The DNA strand which is identical in sequence to the RNA strand is called CODING (sense) strand, and the complementary DNA strand is called the TEMPLATE (antisense) strand. • RNA synthesis is catalyzed by RNA polymerase . Sequences prior to start point are known as “Upstream sequences ” and after start point is known as “ Downstream sequences”
- 2. Major forms of RNA: i.mRNA (messenger RNA) It carries the genetic information that will be expressed ultimately as proteins. Prokaryotic mRNAs are short lived, whereas eukaryotic mRNAs are more stable because of several modifications. ii.tRNA (transfer RNA) It is the adaptor molecule which recognizes the codons of the mRNA on one hand and are covalently bonded to the appropriate amino acid on the other side. iii.rRNA (ribosomal RNA They are found in ribosomes. Prokaryotic ribosomes have three rRNA (23S, 16S, 5S). Eukaryotic ribosomes have 4 rRNA (28S, 18S, 5.8S and 5S).
- 3. Phases of transcription A.INITIATION B.ELONGATION C. TERMINATION • RNA is synthesized under the direction of DNA by the enzyme RNA polymerase ATP, GTP, CTP, and UTP are used to produce an RNA complementary to the DNA template. • RNA synthesis, like DNA synthesis, proceeds in a 5 to 3 ′ ′ direction with new nucleotides being added to the 3 end of ′ the growing chain at a rate of about 40 nucleotides per second at 37°C.
- 4. RNA polymerase In early 1960s, RNA polymerases were discovered in animals, plants and bacteria (Charles Loe, Audrey Stevens, Jerard Hurwitz and Severo Ochoa independently). The fully active form of E. coli RNA polymerase (holoenzyme) is a large multi- subunit protein (430 kD), that has 6 subunits(α2ββ’ωσ). RNA polymerase can initiate chain growth and no primer is needed, unlike DNA polymerase. The core enzyme is composed of five chains and catalyzes RNA synthesis. The sigma factor has no catalytic activity but helps the core enzyme recognize the start of genes. When sigma is bound to core enzyme, the six-subunit complex is termed RNA polymerase holoenzyme.
- 5. Subunits of RNA polymerase and their functions
- 6. 1.A 6 bp sequence centered around 10 bp upstream (-10) of transcription start site. The sequence was initially called Pribnow box, however now it is referred as ‘-10’ region. TATAAT: ‘-10 bp conserved sequence’. The AT rich region melts during the early stage of transcription initiation. 2.A 6 bp sequence around 35 bp upstream (-35) of transcription start site. TTGACA: ‘-35 bp conserved sequence. 3. The distance between the two conserved promoters (between -10 to -35) is around 17±1 bp. Note: the conserved sequences at -35 bp and -10 bp are called Consensus sequences. E. coli E. Coli promoters
- 7. The prokaryotic promoter consensus sequence
- 8. Transcription Unit: is a stretch of nucleotides in DNA that is transcribed into a single functional RNA molecule. It is called an operon in prokaryotes and a gene in eukaryotes. A typical transcription unit has essential parts: A promoter region A start point or initiation sequence A coding segment A terminator sequence RNA polymerase binds to the promoter and transcription starts from the start point in 5,-3’ direction and ends at terminator. Binding of RNA polymerase to promoters: RNA polymerase holoenzyme binds loosely to DNA at first (non-specifically), scanning for the promoter elements. The complex with holoenzyme loosely bound at the promoter is called a Closed promoter complex. The holoenz can melt a shorter local region of DNA (bubble) at the promoter to form an Open promoter complex. The sigma subunit determines the specificity of transcription, by selecting the right promoter. A transcribing complex consists of 3 components and forms the Ternary complex: i.RNA polymerase enzyme ii.DNA molecule containing 12-17 bp transcription bubble. This exposes the template strand to allow it to be copied. iii.A nascent or newly synthesized RNA molecule.
- 9. The length of RNA-DNA hybrid is 8-9 bp approx. As there is no nucleus in prokaryotes, the process of transcription is coupled with translation. As soon as RNA is synthesized it is available for protein synthesis. In eukaryotes, mRNA needs to be transported to cytoplasm before translation can initiate.
- 10. The Initiation of Transcription in Bacteria. The sigma factor of the RNA polymerase holoenzyme is responsible for positioning the core enzyme properly at the promoter. Sigma factor recognizes two regions in the promoter, one centered at 35 and the other centered at 10. Once positioned properly, the DNA at the 10 region unwinds to form an open complex. The sigma factor dissociates from the core enzyme as it begins transcribing the gene.
- 12. • Once bound to the promoter site, RNA polymerase is able to unwind the DNA. • The -10 site is rich in adenines and thymines, making it easier to break the hydrogen bonds that keep the DNA double stranded; when the DNA is unwound at this region, it is called open complex. • A region of unwound DNA equivalent to about two turns of the helix (about 16–20 bases pairs) becomes the “transcription bubble,” which moves with the RNA polymerase as it proceeds to transcribe mRNA from the template DNA strand during elongation. • Within the transcription bubble, a temporary RNA:DNA hybrid is formed. As the RNA polymerase progresses in the 3 to 5 direction along the DNA template, the sigma ′ ′ factor soon dissociates from core RNA polymerase and is available to aid another unit of core enzyme initiate transcription. • The mRNA is made in the 5 to 3 direction so it is complementary and antiparallel ′ ′ to the template DNA. • As elongation of the mRNA continues, single-stranded mRNAi s released and the two strands of DNA behind the transcription bubble resume their double helical structure.
- 13. α subunit: involved in several activities including assembly of the core enzyme. β and β ’ subunits : involved in DNA binding and in phosphodiester bond formation. At each step, RNA polymerase has 3 choices: Elongation: by formation of new phosphodiesterbond at the speed of 50-90 nts/sec Back-tracking: moving backward on the DNA template with dissociation of nascent mRNA. Dissociation: complete dissociation of the complex. Back-trackingis a mean to permit proof-reading of the newly synthesized mRNA. If error is introduced during synthesis, enzyme pauses, back tracks and removes short oligonucleotide (from 3’ end of RNA) containing the error through an endogenous exonuclease activity that is stimulated by the presence of GreA and GreB. These two factors stimulates the inherent Rnase activity of the polymerase. GreA produces short RNA fragments (2-3 nucleotide) and can prevent transcription arrest, but cannot reverse any arrest. GreB produces RNA fragments upto18 nucleotide long and can reverse arrested transcription. These editing methods are known as Hydrolytic editing.
- 14. • Termination of transcription occurs when the core RNA polymerase dissociates from the template DNA. • The sequences within procaryotic terminators often contain nucleotides that, when transcribed into RNA, form hydrogen bonds within the single- stranded RNA. • This intrastrand base pairing creates a hairpin-shaped loop-and-stem structure. This structure appears to cause the RNA polymerase to pause or stop transcribing DNA. • There are two kinds of terminators. Intrinsic or rho-independent termination This type of terminator contains a U-rich sequence downstream from a stretch of nucleotides that can form a stem-loop and stem structure. Formation of the stem loop in the newly synthesized RNA causes RNA polymerase to pause. This pausing is stabilized by the NusA protein. The U-A bonds in the uracil-rich region are not strong enough to hold the RNA and DNA together. Therefore, the RNA, DNA, and RNA polymerase dissociate and transcription stops.
- 15. Intrinsic Termination of Transcription.
- 16. Extrinsic or rho-dependent termination • The second kind of terminator lacks a poly-U region, and often the hairpin; it requires the aid of a special protein, the rho factor(P). • It is thought that rho binds to mRNA and moves along the molecule until it reaches the RNA polymerase that has halted at a terminator. • The rho factor, which has hybrid RNA:DNA helicase activity, then causes the polymerase to dissociate from the mRNA, probably by unwinding the mRNA DNA complex.
- 18. Termination of transcription in E. coli.
- 20. TRANSCRIPTION IN EUKARYOTES There are three major RNA polymerases, not one as in Bacteria. Unlike bacterial polymerase, RNA polymerase II requires extra transcription factors to recognize its promoters.
- 21. polymerase transcribing complex with the pathway of the nucleic acids and some of the more important parts shown. The enzyme is moving from left to right and the DNA template strand is in blue. A protein “wall” forces the DNA into a right-angle turn and aids in the attachment of nucleoside triphosphates to the growing 3 end of the RNA. The ′ newly synthesized RNA (red) is separated from the DNA template strand and exits beneath the rudder and lid of the polymerase protein.
- 22. Unlike bacterial polymerase, RNA polymerase II requires extra transcription factors to recognize its promoters. The polymerase binds near the start point; the transcription factors bind to the rest of the promoter. Three of the most common are the TATA box (located about 30 base pairs before or upstream of the start point), and the GC and CAAT boxes located between 50 to 100 base pairs upstream of the start site The TATA Box and Other Elements of Eucaryotic Promoters.
- 24. Initiation of Transcription in Eucaryotes. The TATA box is a major component of eucaryotic promoters. TATAbinding protein (TBP), which is a component of a complex of proteins called TFIID (transcription factor IID), binds the TATA box.
- 25. • The TFIID transcription factor plays an important role in transcription initiation in eucaryotes. • This multiprotein complex contains the TATA-binding protein (TBP). TBP has been shown to sharply bend the DNA on attachment. This makes the DNA more accessible to other initiation factors. • Introns are removed from the initial RNA transcript (also called pre-mRNA or primary transcript) by a process called RNA splicing. • The intron’s borders are clearly marked for accurate removal. Exon-intron junctions have a GU sequence at the intron’s 5′ boundary and an AG sequence at its 3 end. ′ • These two sequences define the splice junctions and are recognized by special RNA molecules. • The nucleus contains several small nuclearRNA (snRNA) molecules, about 60 to 300 nucleotides long. These complex with proteins to form small nuclear ribonucleoprotein particles called snRNPs or snurps. Some of the snRNPs recognize splice junctions and ensure splicing accuracy. • Splicing of pre-mRNA occurs in a large complex called a spliceosome that contains the pre-mRNA, at least five kinds of snRNPs, and non-snRNP splicing factors. • a few rRNA genes also have introns. Some of these pre-rRNA molecules are self- splicing, ribonuclease P, which cleaves a fragment from one end of pre-tRNA, contains a piece of RNA that catalyzes the reaction.
- 26. Eucaryotic mRNA Synthesis. The production of eucaryotic messenger RNA. The 5 cap is added shortly ′ after synthesis of the mRNA begins.