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Transcription (Introduction, transcription unit, substrates required, Prokaryotic transcription mechanism, Eukaryotic transcription mechanism).pptx
This PPT contains information about, Introduction Transcription unit Prokaryotic transcription Substrates needed for prokaryotic transcription RNA polymerase, Prokaryotic promoter Mechanism of prokaryotic transcription Abortive initiation Eukaryotic transcription Substrates needed for eukaryotic transcription Eukaryotic promoter, transcription factors, activators, co-activators, enhancers, insulators Mechanism of eukaryotic transcription Difference between prokaryotic and eukaryotic transcription
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Transcription (Introduction, transcription unit, substrates required, Prokaryotic transcription mechanism, Eukaryotic transcription mechanism).pptx
- 1. Rashmi M G Transcription 1.Introduction 2. Transcription unit 3. Prokaryotic transcription 4. Substrates needed for prokaryotic transcription 5. RNA polymerase, Prokaryotic promoter 6. Mechanism of prokaryotic transcription 7. Abortive initiation 8. Eukaryotic transcription 9. Substrates needed for eukaryotic transcription 10. Eukaryotic promoter, transcription factors, activators, co- activators, enhancers, insulators 11. Mechanism of eukaryotic transcription
- 2. Rashmi M G Transcription: •Process of formation of transcript (RNA) • Takes place by usual process of complementary base pairing, catalyzed and scrutinized by enzyme- RNA polymerase • It occurs unidirectionally in which RNA chain (transcript) is synthesized from the 5’ to 3’ direction Source: Snustad, Simmons (2012), Principles of genetics, John Wiley& Sons, Inc, Sixth edition, Page no. 257
- 3. Rashmi M G Transcriptionunit: • It is a transcribed segment of DNA • Transcription is a selective process which starts from transcription start site and terminates at transcription termination site • The immediate product of transcription is called primary transcript In eukaryotes, a transcription unit typically carries the information of just one gene and it is termed as monocistronic transcription unit In prokaryotes, a set of adjacent genes is often transcribed as unit termed as polycistronic transcription unit Simple unit Complex unit Simple unit Complex unit Produces primary transcript primary transcript Produces Processed to yield Single type of mRNA, encoding a single protein Processed in more than one way to produce More than one type of mRNA, encoding more than one type of polypeptide
- 4. Rashmi M G Source:Benjamin A Pierce, Genetics, A conceptual approach, Page no. 360 • During transcription only one strand of the transcription unit is transcribed • Therefore, the transcript is identical in sequence with one strand of the DNA which is called the coding strand (also called sense(+) strand) and complementary to the other strand is called template strand (also called antisense (-) strand) • Transcription starts from the first base pair that is called the transcription start site • From this site, RNA polymerase moves along the template, synthesizing RNA until it reaches a termination site • Sequences prior to the transcription start site are described as upstream to it • Sequence after the start site are termed as downstream of it Source: Benjamin A Pierce, Genetics, A conceptual approach, Page no. 358
- 5. Rashmi M G Substratesneeded for transcription: 1. DNA dependent RNA polymerase -RNA polymerase • DNA dependent RNA synthesis catalyzed by enzyme DNA dependent RNA polymerase (simply called RNA polymerase) • Single type of RNA polymerase is responsible for the synthesis of all different types of RNA such as mRNA, rRNA and tRNA • RNA polymerase is a multiunit enzyme made up of 5 difference polypeptides- α,β,β’,⍵,𝜎 Subunits Gene Function α rpoA Assembly of the core enzyme and promoter recognition β rpoB Catalytic center β’ rpoC Catalytic center ⍵ rpoZ Assembly of RNA polymerase 𝜎 rpoD Promoter recognition and transcription initiation Source: Benjamin A Pierce, Genetics, A conceptual approach, Page no. 362 Prokaryotic transcription
- 6. Rashmi M G2. RNA dependent RNA polymerase: • Some viruses like f2 and R17 contain RNA genomes • ssRNA are replicated in the host cells by action of enzymes called RNA-dependent RNA polymerase/ RNA replicases • RNA replicases are specific for RNA of their own virus • RNAs of host cell are not replicated 3. Prokaryotic promoter • Promoter is a cis acting, position dependent DNA sequence necessary for accurately and efficiently initiating transcription of the gene • DNA sequence of the promoter region is recognized by the RNA polymerase • In E coli, the promoter region contains two 6-basepairs of consensus sequences (-10 and -35 sequence). The -10 sequence is also called Pribnow box • The term consensus sequence- the sequence that reflects the most common base or amino acid at each position when a series of related nucleic acid or protein sequences are compared • The -10 sequence= TATAAT consensus; -35 sequence= TTGACA • Some strong promoters have AT rich sequence called UP element-(Upstream Promoter Element) which interacts with the αsubunit of the RNA polymerase Source: Benjamin A Pierce, Genetics, A conceptual approach, Page no. 363
- 7. Rashmi M G Typesof Promoter on the basis of nature of consensus sequences Strong promoter Supports high rate of transcription Weak promoter Low rate of transcription
- 8. Rashmi M G Processof transcription Transcription reaction has 3 stages: Initiation RNA polymerase molecule binds to a promoter sequence on the DNA double helix Elongation Polymerase moves along the template strand, extending its growing RNA chain in the 5’ to 3’ direction by step wise addition of ribonucleotides Termination RNA polymerase reaches termination signal point at which the newly synthesized RNA chain and the polymerase are released from the DNA
- 9. Rashmi M G Source:Snustad, Simmons (2012), Principles of genetics, John Wiley& Sons, Inc, Sixth edition, Page no. 263, 264
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- 11. Rashmi M G Initiation: Itis the step where formation of a first phosphodiester bond between two nucleotides in an RNA takes place RNA polymerase binds at the promoter and starts initiation reaction by forming a closed binary complex Sigma factor confers the ability to recognize consensus sequence within the promoter It also changes the DNA binding properties of RNA polymerase so that its affinity for promoters is increased and reduced for other sequences of DNA Closed complex is converted into an open complex by melting of a short region of DNA within the sequence bound by the enzyme Transcription bubble is created by a local unwinding that begins at the site bound by RNA polymerase Incorporation of first two nucleotides, then phosphodiester bond formation between them. This generates a ternary complex that contains RNA, DNA and Enzyme A stable Ternary complex is only formed when polymerase manages to make an RNA longer than 10 bp
- 12. Rashmi M G Source:Benjamin A Pierce, Genetics, A conceptual approach, Page no. 365
- 13. Rashmi M G Thesigma factor is usually released when the RNA chain reaches 8-9 bases, leaving the core enzyme to undertake elongation The holoenzyme only can initiate transcription, the core enzyme has the ability to synthesize RNA on a DNA template The Holoenzyme remains at the promoter, while it synthesizes the first ~9 nucleotide bonds RNA synthesis is frequently aborted after usually 2 or 3, but upto 10 nucleotides have been joined- this phenomenon is known as abortive initiation The initiation step ends when the enzyme succeeds in extending the chain and clears promoter
- 14. Rashmi M G Source:Peter J Russell, iGenetics A molecular approach, 3rd edition, Page no.85 Image showing the release of sigma factor, leaving the core enzyme to undertake elongation Ternary complex (Holoenzyme+ RNA+DNA)
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- 16. Rashmi M G Elongation: Involvesmovement of transcription bubble by a disruption of DNA structure. The enzyme moves along the DNA and extends the growing RNA chain Enzyme moves, it unwinds the DNA helix to expose a new segment of the template in single stranded condition Nucleotides are covalently added to the 3’ end of the growing RNA chain, forming an RNA-DNA hybrid During each nucleotide addition, the β and γ phosphates are removed from the incoming nucleotide and the hydroxyl group is removed from the 3’ carbon of the nucleotide present at the end of the chain During elongation when RNA polymerase transcribes DNA, unwinding and rewinding occurs As RNA polymerase moves forward along The double helix, it generates positive supercoils ahead and leaves negative supercoils behind Enzyme Gyrase and Topoisomerase I participates during this process Gyrase- removes positive supercoil and introduce negative supercoils Topoisomerase I- removes negative supercoils that develop behind
- 17. Rashmi M G Ternarycomplex (Holoenzyme+ RNA+DNA) Source: Benjamin A Pierce, Genetics, A conceptual approach, Page no. 365
- 18. Rashmi M G Source:Peter J Russell, iGenetics A molecular approach, 3rd edition, Page no.83
- 19. Rashmi M G Termination: Itinvolves recognition of the point at which no further bases should be added to the chain The sequence of DNA required for these reactions is called the terminator The enzyme stops adding nucleotides to the growing RNA chain, releases the completed product and dissociates from the DNA template Bacteria uses 2 distinct strategies for transcription termination Intrinsic termination Rho –dependent termination • Promote dissociation of the polymerase by destabilizing the attachment of the growing transcript to the template • Transcript forms a hairpin structure by forming a complementary base pairing • Intrinsic terminators include G,C rich inverted repeats • Inverted repeats allow the formation of hairpin structure in the transcript which is followed by U- Rich region • Requires the activity of a protein called Rho • Rho is an ATP dependent RNA- stimulated helicase that disrupts the nascent RNA-DNA complex • It binds to RNA at rut site and translocates along RNA in 5’ to 3’ direction until it reaches the RNA- DNA hybrid in RNA polymerase, where it releases the RNA from the DNA • The rut site is rich in C residues and poor in G residues
- 20. Rashmi M G Source:Benjamin A Pierce, Genetics, A conceptual approach, Page no. 366,367
- 21. Rashmi M G Source:Snustad, Simmons (2012), Principles of genetics, John Wiley& Sons, Inc, Sixth edition, Page no. 266
- 22. Rashmi M G Eukaryotictranscription Substrates needed for transcription: 1. Nuclear RNA polymerase • 3 different RNA polymerases for transcription in Eukaryotes: • RNA polymerase I • RNA polymerase II • RNA polymerase III • An additional RNA polymerase is found in mitochondria as well as in the chloroplast, which carry a small DNA molecule of their own • RNA polymerase I: Synthesizes most of the rRNA • RNA polymerase II: Synthesizes mRNA and most of snRNA • RNA polymerase III: Synthesizes a variety of small stable RNAs including tRNA, 5S rRNA and U6 snRNA • All eukaryotic RNA polymerases are large proteins, appearing as aggregates of >500kDa • Each RNA polymerase is a multi subunit protein • RNA polymerase II has a series of heptad repeats with the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser at the carboxyl terminal of the largest pol II subunit. This Carboxyl Terminal Domain (CTD) is both a substrate for several Kinases including the kinase component of TFIIH and a binding site for a wide array of proteins Source: Snustad, Simmons (2012), Principles of genetics, John Wiley& Sons, Inc, Sixth edition, Page no. 272
- 23. Rashmi M G •In addition to 3 RNA polymerases, Plant genomes encode two additional RNA polymerases: • RNA polymerase IV • RNA polymerase V • These polymerases are required for the biogenesis of siRNAs or function of siRNAs in the siRNA-directed DNA methylation pathway, in which siRNA direct the de novo cytosine methylation of complementary DNA sequences • RNA polymerase IV is required for siRNA biogenesis • RNA polymerase V transcripts generate the target for RNA-directed methylation • Mitochondrial RNA polymerase is encoded by nuclear genes • In chloroplasts of higher plants, there are 2 types of RNA polymerases: • Chloroplast –encoded RNA polymerase • Nuclear encoded RNA polymerase
- 24. Rashmi M G 2.Eukaryotic promoter • Promoter = all sequences important in the initiation of transcription of a gene • For some genes, these sequences not only include the core promoter (also called the basal promoter), which is the site at which the initiation complex is assembled, but also one or more upstream regulatory promoter elements which lie upstream of the core promoter and regulates transcription • RNA polymerase I promoter • Promoter consists of a core promoter spanning the transcription start point, between nucleotides-45 and +20, and an Upstream Control Element (UCE) About 100 bp further upstream • The core promoter is generally GC rich except a short AT rich sequence around the start point called the Inr Source: Benjamin A Pierce, Genetics, A conceptual approach, Page no. 371
- 25. Rashmi M G •RNA polymerase III promoter • Includes important sequence elements downstream of the transcription start site, within the transcribed region • Type I internal promoter (found in 5S rRNA genes): requires two internal elements for efficient transcription • Type II internal promoter (found in the tRNA genes): consists of 2 highly conserved sequence blocks • Type III upstream promoter (found in the U6 snRNA genes): consists of TATA box, Proximal Sequence Element (PSE), Distal Sequence Element (DSE) Source: Benjamin A Pierce, Genetics, A conceptual approach, Page no. 372
- 26. Rashmi M G• RNA polymerase II promoter • These promoter are variable and can stretch for several kilo bases upstream of the transcription start site • The core promoter consists of two segments: -30 / TATA box and the initiator (Inr) sequence located around nucleotide +1 • Some genes transcribed by RNA polymerase II have only one of these two components of the core promoter and some have neither and called null genes • A few genes have a Downstream Promoter Element (DPE) which has a variable sequence • TATA box and Inr sequence: serve as specific binding sites for general transcription factors • Other cis acting regulatory promoter sequences (located upstream of TATA box): serve as binding sites for a wide variety of regulatory factors that control the expression of individual genes • 2 regulatory elements that are found in many eukaryotic genes are: • CAAT box (recognized by the activators NF-1 and NF-Y) and GC box (recognized by Sp1 activator) • Consensus sequence present in core promoter (TATA box and Inr) : determines the location of transcription start site • Regulatory promoter elements: influences the frequency of initiation Source: Benjamin A Pierce, Genetics, A conceptual approach, Page no. 368
- 27. Rashmi M G Source:Benjamin A Pierce, Genetics, A conceptual approach, Page no. 370
- 28. Rashmi M G •Transcription initiation by RNA polymerase II • Genes transcribed by RNA polymerase II, first binds with the general transcription factor (GTF) • TFIID complex= TATA binding Protein (TBP)+ 12 TBP associated factors (TAFS) • TBP –binds to DNA which makes contact with the minor groove in the region of the TATA box Transcription initiation with RNA polymerase II • Transcription factors • In addition to RNA polymerase Eukaryotic genes require many protein factors called transcription factors to initiate as well as to regulate transcription • These are trans-regulatory, usually a soluble protein, produced by a gene distant from the chromosome region that it affects • It attaches to the DNA at a gene regulatory site and influences the rate of transcription of a specific gene • Types of transcription factors: • Basal (general) transcription factor): TFIID, TFIIA etc- attaches to gene promoters • Regulatory transcription factors: CBF (CAAT Binding Factor), C/EBP (CAAT/ enhancer-binding protein) , CREB (cAMP Response Element Binding Protein), Sp1 (SV40 early and late promoter binding protein 1)- binds to regulatory transcription factors may be repressor or transcriptional activators Source: Benjamin A Pierce, Genetics, A conceptual approach, Page no. 370
- 29. Rashmi M G GTFFunction TFIID (TBP component) Recognition of TATA box and possibly Inr sequence; forms a platform for TFIIB binding TFIID (TAFs) Recognition of the core promoter; regulation of TBP binding TFIIA Stabilizes TBP and TAF binding TFIIB Intermediate in the recruitment of RNA polymerase II; influences selection of the start point for transcription TFIIF Recruitment of RNA polymerase II TFIIE Intermediate in the recruitment of TFIIH; modulates various activities of TFIIH TFIIH Helicase activity responsible for transition from closed to open promoter complex; possibly influences promoter clearance by phosphorylation of the C terminal domain of the largest subunit of RNA polymerase II
- 30. Rashmi M G AfterTFIID attaches to core promoter Formation of pre-initiation complex (PIC) by the attachment of the remaining GTFs GTFs binds to the complex in the order of TFIIA> TFIIB>TFIIF/ RNA polymerase II> TFIIE > TFIIH Assembly of the initiation complex by addition of phosphate groups to the C-terminal domain (CTD) of the largest subunit of RNA polymerase II Once phosphorylated, the polymerase is able to leave the pre- initiation complex and begin synthesizing RNA (Phosphorylation might be carried out by TFIIH) Source: Benjamin A Pierce, Genetics, A conceptual approach, Page no. 369
- 31. Rashmi M G Transcriptioninitiation in eukaryotic cell is more complex process • It requires a presence of transcriptional activator proteins, that binds to specific sequences in DNA • Eukaryotic gene has many activator proteins. Which together determines its rate and pattern of transcription • These gene regulatory proteins helps RNA polymerase, the general transcription factors, chromatin remodeling complex and mediator all to assemble at the promoter • The mediator is a protein complex consisting of about 20 protein subunits and allows the activator proteins to communicate properly with the polymerase II and with the general transcription factors mediator is the general coactivator of RNA pol II mediated transcription
- 32. Rashmi M G Activatorsand co-activators Activator: • DNA binding protein • Stimulates the transcription initiation • Some will recognize upstream promoter elements and influence transcription initiation only at the promoter to which these elements are attached • Other activators target sites within enhancers and influence transcription of several genes at once Co Activator: • Gene regulatory proteins that do not themselves bind DNA but assemble on DNA bound activator proteins • Enhances the accessibility of the template DNA to general transcription factors or specific activators • Divided into 2 classes: • Chromatin modification complexes • Chromatin remodeling complexes • Consists of 2 domains: • DNA binding domains- region of protein- binds DNA and anchors the transcription factor to the proper site on DNA • Activator domain- activates transcription by interacting with other components of the transcriptional machinery, independently stimulates transcription by interacting with other proteins • Examples of activators: acidic domains, glutamine-rich domains, proline rich-domains
- 33. Rashmi M G Longrange regulatory elements Insulators • Specific DNA element • Controls the action of activator • When insulator placed between an enhancer and promoter, it prevents the enhancers from activating the promoter Enhancers: • Found in genes of mammalian cells • Regulatory sequences located farther away from the transcription start site • Also called silencers • These are transcription control elements • They serve to increase the basal level of transcription which is initiated through the core promoter elements • Consists of an array of binding site, for DNA binding transcription factors • It stimulates transcription in a cell-type specific manner • Transcription factors bound to distant enhancers can, thus, work by the same mechanisms as those bound adjacent to promoters
- 34. Rashmi M G Source:Peter J Russell, iGenetics A molecular approach, 3rd edition, Page no.90