Molec Biol of Gene P1.pdf

Systems, Physiology and Anatomy module, Year 1 Dr Talat Nasim Associate Professor of Therapeutics [email protected] My website: https://www.bradford.ac.uk/staff/tnasim 1 Introduction 2  Dr. Nasim’s Lectures  M...

Systems, Physiology and Anatomy module, Year 1 Dr Talat Nasim Associate Professor of Therapeutics [email protected] My website: https://www.bradford.ac.uk/staff/tnasim 1 Introduction 2  Dr. Nasim’s Lectures  Molecular Biology of the Gene  Gene Expression  Nucleic Acids and DNA Replication  Genetic Variation, health and disease 3  Required Reading Lectures: Molecular Biology of the gene, DNA Replication and Gene Expression:  Biochemistry (3rd Edition); Mathews/ van Holde/Ahren. Chapters 4, 26, 27 and 28.  Molecular Biology of the Cell (4th Edition); Alberts/Johnson/Lewis/Raff/Roberts/Walter: Chapters 4, 5 and 6.  DNA replication: Molecular Biology of the Gene (7th Edition), Watson/Baker/Bell/Gann/Levine/Losick; Chapter 9 Genetic Variation:  Genetics and Genomics in Medicine, Strachen/Goodchip/Chinnery: Chapter 4 Techniques including PCR, DNA sequencing, Agarose Gel Electrophoresis, Western Blotting:  Molecular Biology of the Gene (7th Edition), Watson/Baker/Bell/Gann/Levine/Losick; Chapter 7  Human Molecular Genetics (4th Edition), Tom Strachen and Andrew Reed, Chapter 8, section 2  Genetics and Genomics in Medicine, Strachen/Goodchip/Chinnery: Chapter 3 4 Molecular Biology of the Gene Regulation of gene expression 5  Objectives  To understand the regulation of gene expression in humans (eukaryotes)  Stages where gene expression can be regulated at transcription, splicing and translation  Activities (individual and group) 6  Biochemistry Mathews/van Holde/Ahren Chapters: 4, 27, 28 Human Molecular Genetics Tom Strachan and Andrew Read Chapters: 1, 3 Molecular Biology of the Cell Alberts/Johnson/Lewis/Raff/Ro berts/Walter Chapters: 4, 5, 6 7 23 September 2024 DNA Replication  Resources Medical Sciences Eds. Naish and Syndercombe Court Chapter 2: Biochemistry and Cell Biology Chapter 5: Human Genetics 8 23 September 2024 DNA Replication  REMEMBER! Eukaryotic genes are VERY DIFFERENT in both structure and function to Prokaryotic genes! 9  Transcriptional control Processing control (translation) and non-coding DNA Stages where gene expression can be regulated 10 iGenetics, Russell, Benjamin Cummings, 3rd Ed Transcriptional control 11  Genes and intergenic regions non-coding region between genes ‘Junk’ DNA actually needed for organism plasticity, control of gene expression http://www.humanjourney.us/images/genomeTOgenes.jpg 12  Coding gene structure Enhancers Almost all human genes possess a set of similar characteristics  Promoter  Transcriptional ‘start’ and ‘stop’ signal  Exons and introns  Upstream regulatory regions Any essential features missing? 13  Switching a gene on! Promoter  Not all genes are expressed in all cells, all the time  e.g. some genes may be expressed in response to an external stimulus, others during a particular point in the cell cycle, development etc. (timing is important e.g. Drosphila melanogaster embryo development)  How does a cell keep control over which genes are expressed or not? of DNA that · A sequence Proteins bind to , initiating transcription · Found near transcription site 14  Switching a gene on! Transcriptional activators  Gene expression is driven by RNA polymerase II  In order for a gene to be expressed (switched on) a number of DNA-binding proteins called transcription factors bind in and around the promoter region  Gene expression is fine-tuned via the binding of other DNA- binding proteins to distal regions termed upstream enhancer sequences Transcription suppressor/ +ve repressor binds to promotor region Mediator Transcription RNA Pol II complex Factors activators bas TATA DNA ENHANCER // to -25bp 5’ UTR Exon TATA 15  Open chromatin can be involved in Nanscription and splicing N Activators bind to enhancer sequence and increase expression significantly than without them (basal/low expression) TFs bind around bubble 16 http://www.sciencedirect.com/science/article/pii/S1097276512001165 (Huang 2012)  TRANCRIPTION MACHINERY RNA polymerase Transcription creates the mRNAs, the tRNAs and also the ribosomal RNA (rRNA). These molecules are produced by the enzyme RNA polymerase. 1 large RNA = 2 MRNA = S = tRNA + Small ORNA There are three kinds of RNA polymerases: 1) RNA polymerase I - responsible for the production of the large ribosomal RNA 2) RNA polymerase II – responsible for the production of mRNA 3) RNA polymerase III – responsible for the production of tRNA and the small ribosomal RNA molecules  TRANCRIPTION MACHINERY RNA polymerase molecules collide randomly with the DNA in the nucleus and bind with RNA polymerase + opens helix specific DNA sequences called promoters, i.e., the TATA sequence or TATA box). The RNA polymerase then opens up a short length of the DNA double helix exposing a specific section of DNA on each strand. One strands acts as a template for base pairing with the incoming RNA nucleotides  Transcription +ve Transcription bubble File:Simple transcription elongation1.svg RNA Pol II DNA RNA 5' - 3' direction 19  Switching a gene off! Transcriptional repressors Two types: 1. Interacts with activator - binding site next to activator - blocks function or 2. Overlapping binding sites (activator, repressor) - stops activator from binding  e.g. of a transcriptional repressor is the Wilm’s tumour protein  In the developing kidney, the Wilm’s tumour protein bind to the promoter region of the EGR-1 gene (a transcriptional activator), switching off expression  If the gene encoding Wilm’s tumour protein (WT1) is mutated, this leads to uncontrolled expression of EGR-1 and can lead to the development of kidney tumours early in life. -veis considered a tumour suppressor geneEGR-1 EGR-1  In this respect the WT1 gene Wilm’s EGR-1 EGR-1 TP Mediator Transcription RNA Pol II complex Factors TATA DNA ENHANCER // 5’ UTR Exon -25bp 20 WT1 gene  APPLICATION EXERCISE Activity 1: A. Write a piece of DNA which is 15 base pair long. B. Make a RNA molecule out of the above DNA. DNA T C3 T GC + CGT G CAA 5'A + 3 TAGCACGT TACGAAG51 RNAS' vAG CA CGUU A LG AAG3'  Transcriptional control Processing control (translation) and non-coding DNA Stages where gene expression can be regulated 22 iGenetics, Russell, Benjamin Cummings, 3rd Ed  RNA PROCESSING During transcription the 5’ end of of the mRNA molecule is capped by 1) The addition of methylated G nucliotide by removal of a phosphate by a phosphatase, addition of a GMP via a guananyl transferase, and the addition of a mythyl group via a methyl transferease 2) The 3’ end is cleaved at a specific site and a poly – A tail of up to 200 nucleotides is added by poly – A polymerase. Not all of the the codons transcribed into the mRNA are used. DNA consists of non coding regions (introns) and coding regions (exons). The introns are removed by a process called RNA splicing. RNA SPLICING RNA SPLICING ALTERNATIVE SPLICING Translational control 27  The Genetic Code 64 codons - 20 amino acids 3rd base is degenerate Start Start code AUG codes for methionine (Met) – the first amino acid in a polypeptide 28  Translational control  Cytoplasm, mRNA associates with ribosomes which translate the RNA sequence into a polypeptide chain (protein)  Many ribosomes attach to each mRNA (multiple bubbles)  In time mRNA is degraded (half-life)  The half-life of mRNA is another way in which the cell can regulate gene expression levels 29  TRANSLATION INITIATION Occurs when an initiator tRNA carrying a methionine associates with a small ribosomal unit in association with eukaryotic initiation factor 2 ( eIF2). The small ribosomal unit recognises the a 5’ end of a mRNA capped with two additional initiation factors eIF4G and eIF4E, and scans along the mRNA for a start codon (AUG), which then allows the large subunit bind. TRANSLATION ELONGATION TRANSLATION ELONGATION (4 STAGES) 1) An aminoacyl – tRNA binds to the A site and the tRNA molecule at the E site is released 2) Carboxyl end of the polypeptide chain is uncoupled from the tRNA in the P site and joined by a peptide bond to the amino acid attached to the new tRNA molecule in the A site via a peptide transferase enzyme. 3) Large ribosomal sub unit steps one codon along the mRNA 4) Small ribosomal subunit steps one codon along so that the new peptidyl –tRNA in the A site moves to the P site as and the tRNA that occupied the P site of the ribosome moves 1 codon along the mRNA molecule and becomes the new E –site. This generates a new A site. Driven by Elongation factors EF1 and EF2  TRANSLATION TERMINATION Protein synthesis stops when the ribosome encounters a stop codon (UAA, UAG, UGA). Cytoplasmic release factors bind to the stop codon and free the carboxyl end of the growing polypeptide chain.  One gene - one protein hypothesis? Proposed by Beadle and Tatum (1941) DNA makes RNA makes Protein GENE GENE XX PROTEIN X or PROTEIN Xa PROTEIN Xb PROTEIN Xc One gene makes one protein PROTEIN Xd PROTEIN Xe 17,800 genes How can a similar number of genes give a worm and us? 20,000 genes 34  Answer 1 - alternative Splicing! During splicing the exons can be joined together in a variety of ways 35  Isoforms Alternative mRNAs of a single gene (e.g. Casp8) 36 http://www.bioinfo.mochsl.org.br/miriad/gene/CASP8/?sort=mode  SUMMARY/QUESTIONS  What is an eukaryotic gene?  What is transcription?  What is splicing?  What is translation?  What happens if the transcripts harbour a premature stop signal?  What happens if the transcripts harbour an in-frame stop signal within the coding sequence?  HOME WORK Activity 2: A. Draw a diagram of an eukaryotic gene. B. Write the sequences of that gene (just write the sequence which is around 30 base pair long) C. Make a protein molecule out of the above gene.  The Genetic Code 64 codons - 20 amino acids 3rd base is degenerate Start Start code AUG codes for methionine (Met) – the first amino acid in a polypeptide 39