Transcription in Eukaryotes - PowerPoint Presentation

1. Transcription in Eukaryotes

2. RNA Polymerases of EukaryotesSince typical eukaryotic cells have 10 times as many genes as do bacteria, the whole process of transcription and its regulation is more complex. Indeed, eukaryotes have multiple RNA polymerases, unlike bacteria which have just one. Typical eukaryotes have three different RNA polymerases in the nucleus that transcribe different categories of nuclear genes. In addition, mitochondria and chloroplasts have their own RNA polymerases, which resemble the bacterial enzyme.RNA polymerases I transcribes the genes for the two large rRNA molecules plus 5.8S rRNA and RNA polymerase III transcribes the genes for tRNA, 5S rRNA, and several other small RNA molecules. RNA polymerase II transcribes most eukaryotic genes that encode proteins and as a result is subject to the most complex regulation. Since all types of cells need rRNA and tRNA all the time, RNA polymerases I and III operate constitutively in most cell types.

3. Types of PolymeraseType of PolymeraseResponsible to Produce Location RNA Polymerase IrRNA (18s , 5.8s, 28s)nucleolusRNA Polymerase IImRNA and small nuclear RNAnucleoplasmRNA Polymerase IIItRNA and 5s r RNA nucleoplasm

4. Transcription factors A variety of proteins, known as transcription factors (TF), are also needed for the correct functioning of the eukaryotic RNA polymerases. Transcription factors may be divided into general transcription factors and specific transcription factors. General transcription factors are needed for the transcription of all genes transcribed by a particular RNA polymerase. They are typically designated TFI, TFII, TFIII followed by individual letters, where I, II, and III refer to the corresponding RNA polymerases. Specific transcription factors are needed for transcription of Many transcription factors are involved in controlling gene expression in eukaryotes. Synthesis of rRNA by RNA polymerase I is localized in the nucleolus.

5. Promotor initiates the RNA transcription promoter is a sequence of DNA to which proteins bind that initiate transcription of a single RNA from the DNA downstream of it. This RNA may encode a protein, or can have a function in and of itself, such as tRNA, mRNA, or rRNA. Promoters are located near the transcription start sites of genes (start codon) , upstream on the DNA (towards the 5' region of the sense strand). Promoters can be about 100–1000 base pairs long, class of RNA polymerase recruited to the site.Promoters represent critical elements that can work in concert with other regulatory regions (enhancers, silencers) to direct the level of transcription of a given gene. A promoter is induced in response to changes in abundance or conformation of regulatory proteins in a cell, which enable activating transcription factors to recruit RNA polymerase

6. Eukaryotic promoters complicated and divided into many sub regions 1- regulatory promoter to regulate promoter activity. 2-core promoter: divided into many regionA- recognition element at -35 box (in other reference -30 or -75 )in all cases it is rich with GC nts thus DNA will not be opened here. B- TATA box at -25 box (in other references at -26 or -29 ) as with -10 box in prokaryotic it is rich with AT nts thus the two DNA strand will be opened here (it located 25 nts far away from the start point +1). Discovered by David Hogness in 1977 thus it is called Hogness box. RNA polymerase will bind to -35 box but the two strand is opened here.C-initiator elements (Inr) : represent transcription start point (from -2 to +4) including +1D-Downstream core promoter element (DPE):The DPE is conserved in several eukaryotic organisms and is located at approximately 30 nucleotides downstream of the Transcription Start Site of many TATA-less promoters, acting in conjunction with Transcription Start Site to provide a binding site for TFIID. there are three main recognition sites: TATA box, initiation site sequence(Inr) and DPE, thus highlighting the relevance of this element in promoter activityIn plants, DPE present in different positions and in multiple copies upstream of the Transcription Start Site (TSS). In plants, the upstream region is AT-rich (72.5 %), compared with mammals (52.5 %)

7. Role of TFs in transcription initiation The TATA box is the only element that has a relatively fixed location in relation to the Transcription Start Site. many genes have been found to lack the TATA box In promoters with the TATA box, the transcription factors (TFs) are bind to the DNA of eukaryotic cells to facilitate the binding between the RNA polymerase and DNA, as follows:The TATA box is recognized by the TATA-binding protein (TBP), which is a subunit of TFIID.The binding of TFIID to the TATA box via TATA binding protein is the first step of transcription initiation.TFIIB interacts with TBP as well as the DNA. Thus, these two factors might have an important role in the recognition of the core promoter elements. TFIIB binds to the TFIID(TBP)/TFIIA complex and recruits RNA polymerase II to the promoter, TFIIE and TFIIH bind to the polymerase/promoter complex. the TATA box element was most conserved among all identified elements in both plant and mammalian.

8. Binding of RNA Polymerase II to Promoter Starting with TFIID, which contains TATA binding protein, the components of the TFII complex bind one after another. Finally, TFIIF helps RNA polymerase II to bind to the DNA.

9. Transcription factors regulate the gene expressionTranscription factors bind to either enhancer or promoter regions of DNA adjacent to the genes that they regulate. Depending on the transcription factor, the transcription of the adjacent gene is either up- or down-regulated. Transcription factors use a variety of mechanisms for the regulation of gene expression. These mechanisms include:stabilize or block the binding of RNA polymerase to DNAcatalyze the acetylation or deacetylation of histone proteins. Many transcription factors use one or the other of two opposing mechanisms to regulate transcription:histone acetyltransferase (HAT) activity – acetylates histone proteins, which weakens the association of DNA with histones, which make the DNA more accessible to transcription, thereby up-regulating transcriptionhistone deacetylase (HDAC) activity – deacetylates histone proteins, which strengthens the association of DNA with histones, which make the DNA less accessible to transcription, thereby down-regulating transcription

10. Methylation of CpG sites regulate the gene expressionTranscription factors and methylated cytosines in DNA both have major roles in regulating gene expression. (Methylation of cytosine in DNA primarily occurs where cytosine is followed by guanine in the 5’ to 3’ DNA sequence, a CpG site.) Methylation of CpG sites in a promoter region of a gene usually represses gene transcription, while methylation of CpGs in the body of a gene increases expression.

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12. Enhancers and Mediators control gene expressionSome eukaryotic activators make contact with the general transcription factors However, this is not sufficient to initiate transcription. For this, the mediator is needed. mediator is a protein complex that sits on top of RNA Pol II and provides a site of contact for activators, especially those that are bound at enhancer sequences. One role for the mediator is to recruit and stabilize Pol II at the promoter. The large complex acts as a scaffold, holding the general transcription factors (TFIIB, TFIID, TFIIE, and TFIIH) close to Pol II at the promoter. Two different forms of the mediator have been isolated. The “small mediator complex” is found on actively transcribing genes. The “large mediator complex” is found on genes with Pol II paused at the promoter. This larger complex differs by having an extra component, the mediator kinase, CDK8 (cyclin dependent kinase 8). Thus, removal of CDK8 converts the mediator from a stationary scaffold complex into a transcriptional activator. Enhancers may be found up to several kilobases away from the promoter and either upstream or downstream from the promoters they control. They work by looping the DNA around so that the activator proteins bound at the enhancer can make contact with the transcription apparatus via the mediator complex. This looping mechanism allows a single enhancer to control several genes in its vicinity. The enhancer may located in the intron. That is one reason that introns polymorphisms may have effects although they are not translated. Enhancers can also be found at the exonic region of an unrelated gene and they may act on genes on another chromosome.

13. Activator Proteins and The Mediator The folding of DNA allows numerous activators that are bound to enhancer sequences to approach the transcription apparatus. (B) The mediator complex allows contact of the activators and/or repressors with the RNA polymerase

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15. Releasing of RNA polymerase II and elongation of the RNARelease of RNA polymerase II from the promoter and elongation of the RNA requires three more TFII complexes: TFIIF, TFIIH, and TFIIJ. In particular, TFIIH must phosphorylate the tail of RNA polymerase before it can move. released from the complex of proteins at the promoter and can move forward. All of the TFII complexes except for TFIIH are left behind as RNA polymerase moves forward.The phosphorylation of the RNA polymerase II tail activates the capping enzyme that adds a 7meG-cap onto the 5- end of the mRNA. This cap stabilizes the mRNA and protects against degradation by nucleases

16. RNA Polymerase II Moves Forward From the Promoter Before RNA polymerase II can move forward, the binding of other factors must occur. One of these, TFIIH, phosphorylates the tail of RNA polymerase II. The tail changes position with respect to the body of RNA polymerase II. The other factors leave and RNA polymerase moves along the DNA and begins the process of transcription

17. RNA Elongation After local separation of the strands, the new RNA is synthesized so that it base pairs with one of the DNA strands—the antisense or template strand. The other DNA strand is inactive and is called the sense or coding strand. The enzyme RNA polymerase synthesizes single-stranded RNA in the 5- to 3- direction. The sequence of bases in the RNA is the same as in the sense strand of DNA and complementary to the antisense strand of DNA (except that uracil substitutes for thymine).Ribonucleotides are attached to the OH- molecule on the 3' end of the RNA, transcription always occurs in the 5’3' direction. Multiple RNA polymerases can be active at once, meaning many strands of mRNA can be produced very quickly. RNAP moves down the DNA rapidly at approximately 40 bases per second.RNAP will halts at mismatched base-pairs and correct it.

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20. Terminationin Eukaryotic cells, Termination begins when a polyadenylation signal appears in the RNA transcript. This is a sequence of nucleotides that marks where an RNA transcript should end. The polyadenylation signal is recognized by an enzyme that cuts the RNA transcript nearby, releasing it from RNA polymerase. (as discussed in details in the pervious lecture)

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