Lecture 5 MGD Gene Transcription PDF

Republic of Iraq Ministry of Higher Education and Scientific Stage:S2 Research Module: MGD Wasit Universty College of medicine Lec...

Republic of Iraq Ministry of Higher Education and Scientific Stage:S2 Research Module: MGD Wasit Universty College of medicine Lecture Title: What is a gene and transcription Lecturer Name: Dr –Dhamyaa K. Kadhim The Central Dogma of Molecular biology The Central Dogma: is The flow information from DNA to RNA to Protein in all organism, with exception of Some viruses have RNA as the repository of their genetic information.  Gene expression Gene expression: is the process by which the genetic code (the nucleotide sequence) of a gene is used to direct protein synthesis and produce the structures of the cell. Gene expression involves two main stages: 1-Transcription: Transfer of genetic information from the base sequence of DNA to the base sequence of RNA, mediated by RNA synthesis that occur at nucleus. 2-Translation: Conversion of information encoded in the nucleotide sequence of an mRNA molecule into the linear sequence of amino acids in a protein that occur at cytoplasm.  What is the Gene? The gene: is the basic physical and functional unit of heredity. It consists of a specific sequence of nucleotides at a given position on a given chromosome that codes for a specific protein (or for an RNA molecule). Human carrying between 20000-25000 genes that encoded for all the proteins. These protein–coding genes make up 1–2% of the human genome and transcribed into mRNA. Some other genes produce of other forms of RNA: including transfer RNA (tRNA) and ribosomal RNA (rRNA) involved in Translation. Gene regulation: regulate the gene expression ❖ Each cell in our body has the same protein –coding genes (the same genotype) but not all these genes are expressed in every cell. In fact, in a given cell, almost all genes are switched off most of the time and only about 5% to 10% of the genes in most cells are active. ❖ Liver cells, for example, do not express the genes for eye color, and brain cells do not make enzymes that help digest food. ❖ The process of turning genes on and off is called gene regulation. ❖ So, different cell types use different genes to expresses different proteins (different phenotype ) making them to differ from each other.  Review the RNA structure RNA: is polymer of ribonucleotides covalently linked by 3' →5' phosphodiester RNA is single strand that has direction from 5 ' 3‘ and Bases sequence always written from 5'-end to 3'-end 5'- AGCU-3' Phosphodiester bonds can be cleaved hydrolytically by chemicals, or hydrolyzed enzymatically by nucleases (ribonucleases): ▪ Endonucleases cut the sugar-phosphate backbone within the sequence, either non-specifically or in a sequence-specific manner (ie at a particular site or sites along the strand). ▪ Exonucleases remove one nucleotide at a time from the ends of the molecule, either in a 5’-specific manner or from the 3’ end.  Gene Structure A typical eukaryotic gene consists of the following regions: 1-Transcribed region: involved exons and introns This region contains the DNA sequence that is transcribed into mRNA 2-Regulatory regions (Gene control regions): involved Promoter and enhancer and response elements These regions regulate the transcription of gene  Gene Structure 1-Transcribed region: Exons: is characterized by the following: Code for amino acids and collectively determine the amino acid sequence of the protein product Present in final mature mRNA molecule Numbered from 5'-end of the gene: exon 1, exon 2, etc. Exon 1 at 5'-end of the gene has Untranslated region (5'UTR) and coding region that began with initiation codon (ATG) specify methionine. Last exon at 3'-end of the gene has coding region ends with stop codon(TAA,TAG,TGA) that do not specify any amino acid and Untranslated region (3'UTR). 5'UTR is leader of mRNA strand and 3'UTR is tailing. Mutations in the exons may usually lead to abnormal protein.  Gene Structure 1-Transcribed region: Introns: is characterized by the following Do not code for amino acids Removed (spliced) from the mature mRNA Each intron always began and ends with consensus sequence: GT at 5'-end (5'splice donor) and AG at 3'-end (3'splice acceptor). These are essential for splicing introns out of the primary transcript Mutation at splice sites result in loss of gene production 1-Transcribed region: Gene Structure This region start with the base (py A py) serve as start site (startpoint) for transcription, numbered +1, which is first nucleotide incorporated into the RNA at the 5'-end of the transcript. Subsequent nucleotides in the transcribed region are numbered +2, +3, etc., the direction is called downstream. Regulatory region of the gene, are numbered –1, –2, –3, etc. from the startpoint, the direction is called upstream.  Gene Structure 2-Regulatory regions : Promoter ▪ Consisting of a few hundred nucleotides 'upstream' of the gene (toward the 5' end) that plays a role in controlling the transcription of the gene: determine the startpoint and frequency of transcription by controlling the binding RNA polymerase II  Gene Structure 2-Regulatory regions : Promoter ▪ Has Consensus sequences represent in: 1) TATA box: TATA(A/T)A located -25 region Binds with general transcriptional factors and directs the RNA polymerase II to the correct site (start site) and ensures fidelity of initiation. Mutations at TATA box reduce the accuracy of the startpoint of transcription of a gene. 2) GC-rich regions and CAAT boxes: located region between –40 and –110. Determine how frequently of the transcription event occurs by binding specific proteins. Mutations at these regions reduce the frequency of transcriptional starts 10 to 20 fold. 2-Regulatory regions : Gene Structure Enhancers and response elements Regulate gene expression by binding with specific transcription factors. Enhancers: bind the specific transcription factors (activators or transactivator) that increase the rate of transcription. Silencers or repressor: bind Other specific transcription factors (repressors) that depress the rate of transcription ▪ Enhancers and repressors: found in both upstream and downstream from the transcription site, which located hundreds or even thousands of bases from away the transcription unit. They also function in an orientation-independent fashion.  Transcription Phases of the transcription process: Pre-mRNA Synthesis Initiation – promoter recognition and binding Elongation–the actual process of ‘transcribing’ by RNA polymerase II (5'→3' growing chain): Termination–a sequence-dependent termination of RNA chain growth:  Transcription Initiation: involved Formation of the basal transcription complex as following: The general transcription factors (or basal factors at least six) bind to the TATA box and facilitate the binding of RNA polymerase II. 1) The TATA-binding protein (TBP), a component of TFIID, binds to the TATA box 2) Transcription factors TFII A and B bind to TBP, then RNA polymerase II binds to these factors and to DNA, and is aligned at the startpoint for transcription. 3) Then TFII E, F, and H bind, TFII H acts as ATP-dependent DNA helicase which is unwinding DNA for transcription. This intiation complex can transcribe at a basal level. 4) The rate of transcription can be increased by binding specific transcriptional (transactivators) to the enhancer and they interact with coactivator proteins of TFIID in the complex  Transcription Initiation – promoter recognition and binding  Transcription Elongation–the actual process of ‘transcribing’ by RNA polymerase (5'→3' growing chain): RNA poly II recognize the startpoint and DNA template, RNA poly II reads DNA 3'→5‘ and use the antisense strand (3'→5')o f DNA as a template strand that is copied to produce 5'→3'RNA strand depending on the Watson-Crick complementary. Do not need primer and catalyze 3'→5‘ phosphodiester bond  Transcription Elongation–the actual process of ‘transcribing’ by RNA polymerase (5'→3' growing chain): RNA polymerase II progresses along DNA template leaving complex behind and the initiation complex dissipates upon departure of RNA polymerase. RNA poly has constant synthesis rate about 30-40 nucleotides per second. RNA strand has exactly the same sequence as the DNA 5'→3' sense strand, except that the uracil base instead of thymine.  Transcription Termination–a sequence-dependent termination of RNA chain growth: As RNA polymerase moves along the DNA template, reaching a 3' termination sequence called the polyadenylation signal (AAUAAA). RNA polymerase stops and falls off the DNA template strand. In the process, the pre-mRNA molecule is released and the DNA strands re-form a double helix. Mutation at polyadenylation signal (AAUAAA) will reduce the amount of mRNA.  Transcription Post-transcriptional processing (RNA processing reactions): pre-mRNA (hnRNA)) convert into mature mRNA: 1-Capping – addition of a 5´cap Began immediately after the initiation of RNA synthesis: by adding a methylated guanosine (modified guanosine) to the 5’ end (leader sequence) of the transcript by RNA poly II protects it from degradation by 5’-exonucleases during elongation of RNA chain. increase the efficiency of translation of the mRNA by help the transcript bind to the ribosome during protein synthesis  Transcription Post-transcriptional processing: 2-Tailing (polyadenylation) – addition of a 3´polyA tail Add poly A tail (up to 200 adenine nucleotides) to 3´ terminus by poly A polymerase The poly (A) tail protects the mRNA from degradation by 3' exonucleases. Help in mRNA export: the mature mRNA complexes with poly A-binding protein and other proteins to migrate from nucleus into cytoplasm through nuclear membrane pores. Post-transcriptional processing: Transcription 3-Splicing – the removal of introns; the exons are ‘spliced together’: Introns are removed and exons are spliced together to form the mature mRNA. Split site sequences: beginning (GU/5'splice donor) and ending (AG/ 3'splice acceptor) of each intron, which are essential for splicing introns out of the primary transcript. The exons joined together to form Open Reading Frame (ORF),which is coding area specify amino acids  Gene Expression How can we interpret the presence of 100,000 kinds of mRNA that resulting from25000 genes only? One individual gene can produce different mRNAs coding to different proteins due to: Different splicing products (Alternative splicing): Alternative splicing represent in ability of genes to form multiple processed mRNA contain different combinations of exons that coding to multiple proteins Use of different transcription initiation sites  Messenger RNA mRNA carries information about a protein sequence to the ribosomes, the protein synthesis factories in the cell. It is coded so that every three nucleotides (a codon) correspond to one amino acid. In eukaryotic cells, once precursor mRNA (pre-mRNA) has been transcribed from DNA, it is processed to mature mRNA. This removes its introns—non-coding sections of the pre-mRNA.  Transfer RNA Transfer RNA (tRNA) is a small RNA chain of about 80 nucleotides that transfers a specific amino acid to a growing polypeptide chain at the ribosomal site of protein synthesis during translation. It has sites for amino acid attachment and an anticodon region for codon recognition that site binds to a specific sequence on the messenger RNA chain through hydrogen bonding.  Ribosomal RNA Ribosomal RNA (rRNA) is the catalytic component of the ribosomes. Eukaryotic ribosomes contain four different rRNA molecules: 18S, 5.8S, 28S and 5S rRNA. (single transcription unit (45S) separated by 2 internally transcribed spacers) rRNA molecules are synthesized in the nucleolus. In the cytoplasm, ribosomal RNA and protein combine to form a nucleoprotein called a ribosome. The ribosome binds mRNA and carries out protein synthesis. Several ribosomes may be attached to a single mRNA at any time. rRNA is extremely abundant and makes up 80% of the 10 mg/ml RNA found in a typical eukaryotic cytoplasm. THANK YOU