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Clifton-Bligh, Rory (Dr) Genetic Medicine 2: Principles of molecular genetics
(Problem 1.03, Lecture 10, 2011)
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Principles of molecular biology
genetic paradigm Part 1: DNA and chromosomes Part 2: The language of heredity: from DNA into protein Part 3: Regulation of gene expression Part 4: where things go wrong
The Genetic Paradigm parents transposons miRNAs RNA Reverse transcription protein function children .
The Genetic Paradigm Nucleated cells from the same organism have the same genome Differences in gene expression determines different cellular function The whole organism is the product of interaction between its genes and the environment .
The Genetic Mantra Disease occurs in the presence of an environmental insult and a genetically susceptible individual .
The Genetic Mantra Inheritance: Chromosomal – eg Turners syndrome parents Mendelian RNA Mosaic protein – Autosomal dominant .X-linked .eg diabetes-deafness .eg pseudohypoparathyroidism children Imprinting Uniparental disomy Multigenic .Y-linked function Mitochondrial .eg McCune-Albright .Autosomal recessive .
The Genetic Mantra parents RNA children protein function .
Chemical nature of DNA Nucleic acids contain Compacted in several stages Four different nitrogenous bases ATGC Pyrimidines CT Purines AG Pentose sugar (deoxyribose) phosphate DNA+histones = nucleosomes Nucleosomes+non-histone proteins = chromatin Euchromatin = actively transcribed Heterochromatin = inactive .
Chemical nature of DNA Thymidine HC N C CH3 C C N O H N H C N HC H O H N H N H C N C C CH C N N N C CH C N N H P HC N O H C C N C O H N Adenine Cytosine P Guanine .
Chemical nature of DNA T Thymidine P CTAGGTCATTAAGGCCACGTGCAT A Adenine GATCCAGTAATTCCGGTGCACGTA C Cytosine P Guanine G .
Organisation of DNA CTAGGTCATTAAGGCCACGTGCAT GATCCAGTAATTCCGGTGCACGTA .
The double-helix is the basis of heredity Explains: Replication Inheritance Complementary basepairing also central to understanding RNA. protein .
Organisation of DNA HISTONES .
Summary: part 1 DNA consists of two complementary chains of nucleotides The double-helix is the mechanism for heredity DNA resides in the nucleus (eukaryotes) DNA is packaged in chromosomes Chromosomes contain long strings of genes… .
What is a gene? .
Region of DNA transcribed into RNA Unit of heredity Humans: 30 000-40 000 genes Expressed regions (exons) ie present in mature mRNA Intervening regions (introns) are excised as a new RNA transcript is processed to its mature form Promoter: initiates transcription and regulates efficiency of transcription
The language of the genome
of the promoter
“core” promoter TATA box
Transcription Recognition IIA of the promoter IIF IIB IIE Transcription factors IID Pol II IIH .
... . .GTAC.Transcription RNA polymerase transcribes RNA from DNA Pol II .CAUG..
Transcription RNA transcript undergoes modification CAP AAAAAAAAAAAAAAn Pol II .
Transcription Splicing exons (introns removed) Introns CAP AAAAAAAAAAAAAAn Exons .
Transcription Messenger RNA is exported from the nucleus to cytoplasm CAP AAAAAAAAAAAAAAn .
only one is used .Translation mRNA sequence is decoded in sets of three nucleotides called codons Each codon specifies an amino acid Each mRNA therefore encodes a polypeptide chain Although there are 3 possible “reading frames” for mRNA.
RNA codons GGCCGCAUGAACCGUCGCCCGUCACCGUUAUUGCGU methionine asparagine arginine arginine proline serine proline leucine .
The Genetic Code Redundant ie several nucleotide triplets U may code for one amino acid C Universal A G U Phe Leu Leu Ile Met Val C Ser A Tyr stop stop G Cys Trp Arg Ser Arg Gly stop U C A G U C A G U C A G U C A G Pro His Gln Asn Lys Asp Glu Thr Ala .
Translation The ribosome: Large ribosomal subunit Small ribosomal subunit Transfer RNA Amino acid Anti-codon .
U C Ser A Tyr stop stop G Cys Trp Arg Ser Arg Gly stop U C A G U C A G U C A G G U C A G Translation methionine U C A G Phe Leu Leu Ile Met Val Pro His Gln Asn Lys Asp Glu Thr UAC Ala AUG mRNA .
Translation UAC AUG .
Translation Growing polypeptide chain .
Translation U U C A G Phe Leu Leu Ile Met Val C Ser A Tyr stop stop stop G Cys Trp Arg Ser Arg Gly stop Post-translational modification U C A G U C A G U C A G UAG U C A G G Pro His Gln Asn Lys Asp Glu Thr Ala .
Protein modification The shape of a protein is specified by amino acid sequence Molecular chaperones Hsp60 and hsp70 Prevent protein aggregation Fold protein into correct 3-d form Quality control: ubiquitination Post-translational modifications Disulfide bonds Glycosylation phosphorylation .
G. N Engl J Med 1998.Protein folding Kuznetsov.339:1688-1695 . et al.
Post-translation modification Preproinsulin Proinsulin insulin .
Summary: part 2 A gene is DNA that is transcribed into RNA RNA is complementary to one DNA strand RNA is modified before export from the nucleus mRNA sequence is decoded in triplet nucleotides by ribosomes tRNAs are the bridge between RNA codons and cognate amino acids Proteins are folded and processed to achieve function .
The basic idea… RNA Which protein How much protein function When (timing) In what combination .
modification Amount of a gene expressed Type of gene product expressed .Regulation of gene expression For most genes the most important control point of expression is at the initiation of RNA transcription DNA level: chromatin. promoters. enhancers RNA level: RNA stability Protein level: protein degradation. histones.
Chromatin structure and transcription Ac Histone complex Ac Histones acetylated =transcription facilitated Histones deacetylated =transcription repressed .
Transcription factors can recruit histone modifying enzymes Histone Deacetylase “switch” Histone Acetylases .
Gene regulation by DNA Nuclear or Receptor Transcription Factor .
Nuclear receptor Histone acetylases IIAIIB IIF IID IIE Pol II IIH Other Transcription factors .
E. A.Gene regulation at RNA level Differential splicing can produce… Variant of same protein product Different protein product E1 E1 E2 E2 E3 E3 calcitonin CGRP gene RNA E6 E6 calcitonin CGRP E1 E2 E3 calcitonin E1 E2 E3 CGRP E6 calcitonin Guttmacher. et al.347:1512-1520 CGRP . N Engl J Med 2002.
Summary: part 3 Control of gene expression is mostly at the point of transcription Gene transcription is controlled by combinations of regulatory proteins Each cell type has a specific combination of regulatory proteins that directs gene expression appropriate to that cell Histone modification affects gene expression .
Coding DNA mutations Silent Missense Nonsense Frameshifts .
Coding DNA mutations Silent Normal sequence: …CCG TCA CCG… Serine Silent mutation: …CCG TCC CCG… Serine .
Coding DNA mutations Silent Missense Normal sequence: …CCG TCA CCG… Serine Missense mutation: …CCG CCA CCG… Proline .
Coding DNA mutations Silent Missense Normal sequence: …CCG TCA CCG… Serine Nonsense Missense mutation: …CCG TAA CCG… Stop .
Coding DNA mutations Silent Missense Normal sequence: …CCG TCA CCG… Serine Proline Nonsense Frameshifts Frameshift mutation: …CCG TCG ACC G… Serine Threonine .
Coding DNA mutations Can reduce amount of mRNA (and therefore protein) alter function of protein (eg mutation in catalytic site of enzyme – either activating or inactivating) cause protein mis-folding Can Can .
leading to premature termination of the mRNA and complete absence of CFTR protein. 2004 . frameshift. UpToDate. Defective protein processing – these mutations prevent the protein from trafficking to the correct cellular location. Defective channel function – These mutations lead to diminished channel activity or rate of ion flow when compared to normal CFTR.Cystic fibrosis: mutations in CFTR gene Defective protein production – usually caused by nonsense. or splice-site mutations. delta F508 (deletion of a phenylalanine residue at codon 508). Includes the most common CFTR mutation.
Walter. Raff. 2002.Suggested reading Molecular Biology of the Cell by Alberts. 1992. Genes VII by Benjamin Lewin. Roberts. Johnson. Garland Publishing. A Genetic Switch: Phage and Higher Organisms by Mark Ptashne. Oxford University Press. Lewis. 1999. Inc. Blackwell Science. .. Paul Siliciano. Martin Klotz.
Methylation Methylation of cytosines=silencing Eg X chromosome inactivation Important cause of imprinting Too much or too little methylation can cause imprinted disorders: Beckwidth-Wideman syndrome Angelman syndrome .
Eg glucocorticoid-remediable hypertension is due to fusion of the promoter region of the gene for CYP11B1 and the coding sequences of CYP11B2. resulting in ACTH-dependent activation of the aldosterone synthase Different gene expressed .Promoter mutations Reduce gene expression Eg some cases of alpha and beta thalassemia are due to mutations in promoter or enhancer sequences for the alpha or beta globin genes.
R. C.336:487-491 .Cystic fibrosis: severity correlates with amount of functional CFTR Different mutations variably affect CFTR expression and function Stern. N Engl J Med 1997.
346:45-53 .Protein misfolding Aggregations of misfolded protein can cause disease: Cystic fibrosis α1.A Model for Conformational Diseases. Alpha1-Antitrypsin deficiency. New Engl J Med.antitrypsin deficiency Huntington’s disease Alzheimer’s disease Parkinson’s disease (familial) Amyloid Prion diseases Carrell and Lomas 2002.
Mutations in different regions of the gene can produce different phenotypes Burke. W. N Engl J Med 2003.349:969-974 .
A special kind of insertion… Trinucleotide repeats Found in several genes Number varies among healthy individuals Increase in number of repeats above a critical threshold associated with disease Eg Huntington’s disease .
G. D. N Engl J Med 2002.346:683-693 .22) Leading to the Philadelphia (Ph) Chromosome and the Role of BCR-ABL in the Pathogenesis of CML Savage.Chromosomal translocations can produce new genes Translocation (9. et al.
Splice-site mutations Loss of critical exon gene expression Reduced Eg several common forms of alpha and beta thalassemia .