Eukaryotic Transcription.pptx

Eukaryotic Transcription

1. Eukaryotic Transcription and its Regulation Assignment Presentation on

2. Introduction The process by which RNA molecules are synthesized from DNA template. It is catalysed by the enzyme RNA polymerase (Wiess, 1960). All cellular RNAs are synthesized from the DNA templates through this process. DNA regions that can be transcribed into RNA are called structural genes. Only one strand of the DNA duplex is copied which serves as template and is called antisense strand or template strand (3’-5’). RNA strand has base sequence complimentary to this strand. The other strand of the DNA duplex has sequence same as that RNA molecule and this strand is called sense strand or coding strand (5’-3’).

3. Only the templet strand is used for the transcription, but the coding strand is not. Only a small portion of DNA is transcribed in response to the development requirement, physiological need and environmental changes.

4. Difference between replication and transcription Replication Transcription Template Double strand Single strand Substrate dNTP NTP Primer Yes No Enzyme DNA polymerase RNA polymerase Product dsDNA Ss RNA Base pair A-T,G-C A-U,G-C

5. RNA polymerase An enzyme that catalyze RNA synthesis. It does not need a primer , rather it can initiate transcription denovo . It perform the same reaction in all cells, from bacteria to humans. Bacteria have only a single RNA polymerase Eukaryotes have 3 RNA polymerase i.e . RNA Pol, I,II, III. Pol ll is the most studied of these enzymes, and is responsible for transcribing all protein-encoding genes. Pol l & ll are responsible for transcribing specialized, RNA encoding genes. The shape of RNA polymerase resemble a crab claw.

6. Transcription in Eukaryotic RNAs Eukaryotic transcription proceeds in three sequential stages: initiation, elongation, and termination. The transcriptional machinery that catalyzes this complex reaction has at its core three multi-subunit RNA polymerases. Name Location Function Product RNA Polymerase I Nucleolus Cellular RNA synthesis larger ribosomal RNA ( rRNA ) (28S, 18S, 5.8S) RNA Polymerase II Nucleus Transcribes genes that produce mRNA Messenger RNA (mRNA), most small nuclear RNAs snRNAs ), small interfering RNA (siRNAs) and micro RNA (miRNA). RNA Polymerase III Nucleus Transcribes tRNA and other small RNA genes transfer RNA (tRNA), other small RNAs (including the small 5S ribosomal RNA (5s rRNA), snRNA U6, signal recognition particle RNA (SRP RNA) and the stable short RNAs

7. CORE PROMOTER It refers to the minimal set of sequence elements required for accurate transcription initiation by Pol ll. A core promoter is about 40 nucleotide long , extending upstream of the transcription start site. Relative to the transcription start site, there are 4 elements found in Pol ll core promoter. These are the TFIIB regognition element (BRE), the TATA element, the initiator ( Inr ) & the downstream promoter element ( DPE ). Promoter includes only 2 or 3 of these 4 elements.

8. PRE INITIATION COMPLEX FORMATION The GTFs help polymerase bind to the promoter and melt DNA. The complete set of GTFs & polymerase bound together at the promoter and poised for initiation, is called as pre-initiation complex. Many Pol ll promoters contains TATA elements , where pre-initiation complex formation begins. The TATA elements recognized by GTFs called TFIID. The component of TFIID that binds to the TATA DNA sequence is called TBP . The other subunit is TAEs that control the DNA binding activity of TBP. Sometimes the GTFs are not sufficient to promote significant expression. Rather, the additional factors are required such as mediator complex, DNA binding regulatory proteins and chromatin modifying enzymes.

9. The resulting TBP-DNA complex provide a platform for attachment of other GTFs & polymerase. The factors TFIIA & TFIIB bind to this complex. After that TFIIF together with polymerase also bind to the complex. At last, the two factors TBIIE & TBIIH bind to upstream of Pol. ll resulting in the formation of pre-initiation complex. Formation of this complex containing these all components is followed by promoter melting. Promoter melting in eukaryotes requires hydrolysis of ATP and is mediated by TFIIH. The large subunit of Pol. Ll has a C- terminal domain (CTD). Which extends as a ‘tail’. The CTD contains a series of repeats of heptapeptide sequence: Tyr-Ser-Pro - Thr -Pro-Ser.

10. ELONGATION Once polymerase has initiated transcription, it shifts into the elongation phase. Elongation requires another set of factors, such as TFIIS & hSPT5, known as elongation factors. This factors also favour the phosphorylated form of CTD. The phosphorylation of CTD leads to an exchange of initiation factors with elongation factors. Various proteins are thought to stimulation by Pol ll. The protein P- TEFb stimulate in 3 separate steps. This protein bound to Pol ll and phosphorylates the serine residue at position 2 of the CTD repeats.

11. This P- TEFb also activates another protein, called hSPT5 which is an elongation factor.

12. POLYADENYLATION & TERMINATION Once the elongation is completed, it proceeds through the RNA processing events i.e . polyadenylation and termination. Polyadenylation occurs at the 3' end of the mRNA which is linked with the termination of transcription. The polymerase CTD tail is involved in recuiting the enzymes necessary for poly adenylation. Two protein complexes are carried by the CTD of polymerase called , CPSf (cleavage & polyadenylation specificity factor) & CstF (cleavage stimulation factor) The sequence which one transcribed into RNA , trigger transfer of these factors to the RNA, are called poly-A signals. Once CPSF & CstF bound to the RNA, it result in the RNA cleavage and then polyadenylation.

13. After the cleavage of RNA, polyadenylation is mediated by an enzymes called poly -A polymerase (PAP) followed by the addition of poly-A binding protein. This protein along with the enzyme uses ATP as precursor and adds the nucleotides , using the same chemistry as RNA polymerase. Before termination , the RNA molecule become very long due to addition of several nucleotides. The polymerase along with CPSF & PAP then dissociates from the templates, releasing the new RNA , which is degraded without ever leaving the nucleus. This involves the termination of RNA, i.e. the mature mRNA is released from polymerase and then transported from the nucleus.

14. Polyadenylation & Termination

15. Processing of mRNA 1. 5’ capping 2. Splicing 3. Cleavage & Poly A Tail

16. 5’ Capping The 7-methylguanosine structure is called a type 0 cap and is the commonest form in yeast. In higher eukaryotes, additional modifications occur A second methylation replaces the hydrogen of the 2¢–OH group of what is now the second nucleotide in the transcript. This results in a type 1 cap . If this second nucleotide is an adenosine, then the amino group attached to carbon number 6 of the purine ring might also be methylated. Another 2¢–OH methylation might occur at the third nucleotide position, resulting in a type 2 cap .

17. 3’ Cleavage & Polyadenylation

18. Splicing The excision of introns and the joining together of all the exons in a gene in proper sequence to yield the mature mRNA is called splicing .

19. Alternative Splicing : When a single pre- mRNA molecules is processed in 2 or more ways to yield more than one type of mature m RNA, is called alternate splicing.

20. Its occur in chloroplast . Trans-splicing - Sequences present in 2 or more RNA molecules are spliced together is called trans- splicing.

21. Regulation of transcription Regulation of Transcription in Eukaryotes, Although the control of gene expression is far more complex in eukaryotes than in bacteria, the same basic principles apply. The expression of eukaryotic genes is controlled primarily at the level of initiation of transcription , although in some cases transcription may be reduced and regulated at subsequent steps. As in bacteria, transcription in eukaryotic cells is controlled by proteins that bind to specific regulatory sequences and modulate the activity of RNA polymerase. Combined actions of multiple different transcriptional regulatory proteins. In addition, the packaging of DNA into chromatin and its modification by methylation impart further levels of complexity to the control of eukaryotic gene expression.

22. cis – Actingregulatory sequences: Promoter and Enhancers Transcription in bacteria is regulated by the binding of proteins to cis-acting sequences that control the transcription of adjacent genes. Genes transcribed by RNA polymerase II have two core promoter elements, the TATA box and the Inr sequence, that serve as specific binding sites for general transcription factors. Other cis-acting sequences serve as binding sites for a wide variety of regulatory factors that control the expression of individual genes. These cis-acting regulatory sequences are frequently, though not always, located upstream of the TATA box. For example, two regulatory sequences that are found in many eukaryotic genes were identified by studies of the promoter of the herpes simplex virus gene that encodes thymidine kinase. Both of these sequences are located within 100 base pairs upstream of the TATA box: Their consensus sequences are CCAAT and GGGCGG (called a GC box). Specific proteins that bind to these sequences and stimulate transcription have since been identified.

23. A eukaryotic promoter The promoter of the thymidine kinase gene of herpes simplex virus contains three sequence elements upstream of the TATA box that are required for efficient transcription: a CCAAT box and two GC boxes (consensus sequence GGGCGG). In contrast to the relatively simple organization of CCAAT and GC boxes in the herpes thymidine kinase promoter, many genes in mammalian cells are controlled by regulatory sequences located farther away (sometimes more than 10 kilobases) from the transcription start site. These sequences, called enhancers, were first identified by Walter Schaffner in 1981 during studies of the promoter of another virus, SV40. In addition to a TATA box and a set of six GC boxes, two 72-base-pair repeats located farther upstream are required for efficient transcription from this promoter. These sequences were found to stimulate transcription from other promoters as well as from that of SV40, and, surprisingly, their activity depended on neither their distance nor their orientation with respect to the transcription initiation site. They could stimulate transcription when placed either upstream or downstream of the promoter, in either a forward or backward orientation.

24. The SV40 enhancer . The SV40 promoter for early gene expression contains a TATA box and six GC boxes arranged in three sets of repeated sequences. In addition, efficient transcription requires an upstream enhancer consisting of two 72-base-pair (bp) repeats.

25. Action of enhancers Without an enhancer, the gene is transcribed at a low basal level (A). Addition of an enhancer, E—for example, the SV40 72-base-pair repeats—stimulates transcription. The enhancer is active not only when placed just upstream of the promoter (B), but also when inserted up to several kilobases either upstream or downstream from the transcription start site (C and D). In addition, enhancers are active in either the forward or backward orientation (E). The ability of enhancers to function even when separated by long distances from transcription initiation sites at first suggested that they work by mechanisms different from those of promoters. However, this has turned out not to be the case: Enhancers, like promoters, function by binding transcription factors that then regulate RNA polymerase. This is possible because of DNA looping, which allows a transcription factor bound to a distant enhancer to interact with RNA polymerase or general transcription factors at the promoter.

26. The immunoglobulin enhancer. The immunoglobulin enhancer. The immunoglobulin heavy-chain enhancer spans about 200 bases and contains nine functional sequence elements (E, μ E1-5, π, μ B, and OCT), which together stimulate transcription in B lymphocytes.

27. Relationship between chromatin & transcription Transcription is possible in less condensed region of chromatin i.e Euchromatin Even in less condensed region, the winding of DNA in nucleosome is a major obstacle to transcription This inhibitory effect of nucleosome is relieved by acetylation of histone and by non histone proteins HMG-142, HMG-17 Transcriptionally active chromatin Similarly histone de-acetylases are associated with transcriptional repression

28. Gene Regulation at DNA Level Chromatin Remodeling DNA Methylation Cytosine residue in vertebrate DNA can be modified by the addition of methyl group at the 5-carbon position DNA methylation occurs at CpG position Methylation of C – reduced transcription Methylation of C Methylated DNA Methylated histone binding protein Formation of heterochromatin Transcriptional inactivation.

29. Metal-regulated transcription in eukaryotes The major role metals play in biology is highlighted by the fact that metals compose 17 of the 30 elements known to be essential for life. Metals constitute a nutritionally important, yet cytotoxic component in our environment. A number of metals can be categorized into three groups with respect to these considerations. One group, of which zinc (Zn) is a good example, provides essential cofactors for a wide variety of metalloproteins and enzymes . This metal is normally not toxic except at extremely high concentrations. A second group, of which cadmium (Cd) is a member, constitutes metals which have no known nutritional value yet are highly cytotoxic . The third group is composed of metals such as copper (Cu), which are both essential for life processes as cofactors for many enzymes and are extremely potent cellular toxins . Indeed, because metal concentrations Dennis J. Thiele, 1992

30. Zinc finger domains contain repeats of cysteine and histidine residues that bind zinc ions and fold into looped structures (“fingers”) that bind DNA. These domains were initially identified in the polymerase III transcription factor TFIIIA but are also common among transcription factors that regulate polymerase II promoters, including Sp1. Other examples of transcription factors that contain zinc finger domains are the steroid hormone receptors, which regulate gene transcription in response to hormones such as estrogen and testosterone. The helix-turn-helix motif was first recognized in prokaryotic DNA-binding proteins, including the E. coli catabolite activator protein (CAP). In these proteins, one helix makes most of the contacts with DNA, while the other helices lie across the complex to stabilize the interaction. In eukaryotic cells, helix-turn-helix proteins include the homeodomain proteins , which play critical roles in the regulation of gene expression during embryonic development. Molecular cloning and analysis of these genes then indicated that they contain conserved sequences of 180 base pairs (called homeoboxes) that encode the DNA-binding domains (homeodomains) of transcription factors. In the Leucine zipper structure leucine residues occure every seven amino acids in an α -helical structure such that the leucines occure every two turns on the same side of the helix. Helix-loop-helix motif is now believed to play a similar role to the leucine zipper in mediating protein dimerization and facilitating DNA binding by the adjacent basic DNA binding motif.

31. Regulation of the initiation of eukaryotic transcription. DNA sequences that determine transcriptional regulation of a typical eukaryotic gene consist of a core promoter, which serves as a binding site for the GTF TFIID, and regulatory promoter or enhancer sequences, which bind transcriptional activators. The RNA polymerase II transcription machinery consists of over 50 proteins which are bind to the core promoter in two steps: binding of TFIIA-TFIID , followed by binding of a large pre-assembled holoenzyme complex consisting of the remaining GTFs, RNA polymerase II and associated regulatory proteins . Activators function to increase binding of the transcription machinery to the promoter in at least two ways:( i ) simple protein-protein interactions with activators increases the affinity of the transcription machinery for the promoter, and (ii) some activators stabilize a conformation of the TFIIA-TFIID-DNA complex that enhances binding of the holoenzyme. Recent studies have identified many co-activators that function with activators to increase transcription by the RNA polymerase II transcription machinery. Although some co-activators may serve as bridges to connect activators with the transcription machinery. Grace Gill, 2001

32. Source: Genome 4 by T. A. Brown Davis S. L. (1990). Eukaryotic transcription factores . Biochemical journal, 270 : 281-289 Grace Gill. (2001). Regulation of the initiation of eukaryotic transcription. Essays in biochemistry 37 : 33-43