BHCS2003 DJP L02 The Central Dogma PDF

Summary

These notes cover the central dogma of molecular biology, detailing the processes of replication, transcription, and translation. The document also discusses genes, eukaryote gene structure, and transcription.

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BHCS2003 Genetic Continuity & Diversity The central dogma of molecular biology DNA → RNA → Protein...

BHCS2003 Genetic Continuity & Diversity The central dogma of molecular biology DNA → RNA → Protein Replication: maintain and copy genetic information The Central Dogma Transcription: Information encoded in a gene copied into pre-messenger RNA (pre-mRNA) From DNA to Protein in the nucleus Processed to form mRNA that moves into cytoplasm Translation: conversion of information into linear sequence of amino acids Post-translational modification: folding to form protein 1 2 The central dogma of molecular biology Genes Eukaryote cells contain certain DNA sequences such as A gene is a part of a DNA molecule that serves as a template for LINE-1 repetitive DNA (see later) that encode reverse making a functionally important RNA molecule transcriptases Many different types of RNA molecule, but 2 broad groups These can produce DNA sequences from an RNA template Coding RNA molecules contain a sequence that can be decoded to generate a polypeptide sequence Thus, the central dogma is not strictly valid This is messenger RNA (mRNA) Noncoding RNA molecules do not serve as templates for making polypeptides. Instead, many are involved in regulation of gene expression Regulation can be general, or just target a small set of specific genes. Process may involve catalytic RNA molecules 3 4 Eukaryote gene structure Transcription Transcription is the synthesis of an RNA molecule from a DNA template Requires three main components: DNA template Ribonucleoside triphosphates Generalised structure of a eukaryotic gene to build a new RNA molecule This example has three introns and four exons Transcription apparatus consisting of proteins necessary to catalyse RNA synthesis Here will consider transcription to produce mRNA 5 6 1 DNA Transcription Successive deoxyribose units are linked Transcription: the synthesis of an RNA molecule from a DNA template through carbon atoms labelled 5’ and 3’ Proceeds in 5’ → 3’ direction, RNA polymerase adds nucleotides to the Chains in a DNA double helix are anti-parallel 3’ end of a polynucleotide If a DNA double helix is drawn vertically, one Convention to write DNA and RNA sequences in 5’ to 3’ direction: chain will have 5’ end at top, the other 3’ end AGTTGCACG means 5’- AGTTGCACG -3’ Same true of DNA-RNA double helices that are temporary intermediates when a gene is Only one strand of DNA transcribed – the template strand transcribed RNA transcript is complementary to this (T where template has A, C Enzymes that act on 5’ end cannot act on 3’ where template has G, etc) end and vice-versa Remember, strands are anti-parallel and RNA uses U instead of T, so Several important consequences writing the sequence in 5’ → 3’ direction gives: CGUGCAACU 7 8 Transcription Consensus sequences Remember, strands are anti-parallel and RNA uses U instead of T, so Consensus sequence: writing the sequence in 5’ → 3’ direction gives: consists of most commonly encountered bases at each CGUGCAACU position in a group of related sequences Relationship is not immediately obvious even with a short sequence To avoid problems, by convention, the DNA sequence of a gene is written as the complementary strand which is called the coding strand or A consensus sequence in a sense strand: set of nucleotides usually implies an association with CGTGCAACT an important function RNA sequence same as sense strand apart from using U for T The DNA template strand is also known as the antisense strand 9 10 RNA polymerases Transcription Unit Most eukaryotes have three types of RNA polymerase Transcription unit: a stretch of DNA that encodes an RNA responsible for transcribing different classes of RNA molecule plus the sequences needed for its transcription RNA polymerase I Within a transcription unit are three critical regions: transcribes large rRNAs Promoter: a DNA sequence that transcription apparatus RNA polymerase II recognises and binds to. Indicates which DNA strand is to pre-mRNA, some snRNAs, snoRNAs, some miRNAs be transcribed and the transcription start site RNA polymerase III tRNAs, small rRNAs, some snRNAs, some miRNAs RNA-coding region: a sequence of DNA nucleotides copied into an RNA molecule RNA polymerase I, II, and III recognise different promoters Terminator: a sequence of nucleotides that signals where RNA polymerases do not require primers transcription is to end 11 12 2 Promoters Promoters and RNA polymerase II Promoter recognition is carried out by accessory proteins that bind to Core promoter located upstream of gene and site to which basal the promoter and recruit a specific RNA polymerase to the promoter transcription unit binds Many proteins needed for binding of RNA polymerases to DNA Includes one or more consensus sequences such as the TATA box with templates and different promoters require different proteins the consensus sequence TATAAA This and other consensus sequences in the core promoter are One class of accessory proteins are general transcription factors which recognised by transcription factors that bind to them and act as a with RNA polymerase form the basal transcription apparatus platform for assembly of the basal transcription apparatus Assembled near the start site and initiates minimal level of transcription Regulatory promoters located upstream of core promoter Another class are transcriptional activator proteins which bind to A variety of different consensus sequences specific DNA sequences and bring about higher levels of transcription by stimulating assembly of basal transcription apparatus Activator proteins bind to these and make contact with the basal transcription apparatus to affect transcription rate Other activator proteins also regulate transcription by binding to more distant sequences called enhancers 13 14 Promoters From DNA to chromosomes Promoters of genes transcribed by RNA polymerase II consist of a core promoter and a regulatory promoter that contain consensus sequences Not all consensus sequences are found in all promoters Chromosome 17 as seen in a G-banded, 400 band preparation 15 16 Transcription process Initiation DNA is complexed with histone proteins in highly Assembly of transcription machinery on the promoter compressed chromatin and must be modified before transcription can begin (see later) Consists of polymerase II and series of transcription factors Chromatin modification results in a more open structure so Forms a complex consisting of more than 50 polypeptides that DNA can be accessed by the transcription machinery After chromatin modification, the basal transcriptional The process of transcription itself consists of three stages: apparatus is recruited to the core promoter Initiation The basal transcriptional apparatus consists of general Elongation transcription factors and a protein complex, the mediator Termination General transcription factors include TFIIA, TFIIB, TFIID, etc 17 18 3 Initiation Initiation A first step in initiation is binding of TFIID to the TATA box After assembly, conformational changes cause 10 – 15 bp DNA at transcription start site to separate which produces TFIID consists of several polypeptides, one is the TATA- the single-stranded DNA that will act as a template binding protein (TBP) which recognises and binds to the TATA consensus sequence, bending the DNA and partly This template is positioned within the active site of RNA unwinding it polymerase creating a structure called the open complex Synthesis of RNA begins as phosphate groups are cleaved TBP binds to the minor groove of off nucleoside triphosphates and resulting nucleotides are DNA straddling the double helix joined together to form an RNA molecule Other transcription factors bind to additional consensus sequences in core promoter and to RNA polymerase and position the polymerase over the transcription start site. 19 20 Elongation Elongation After about 30bp synthesised, the RNA polymerase leaves Bends at a right angle the promoter and process enters elongation stage positioning ends of DNA- RNA hybrid at the active Many transcription factors left behind and can be used to site of the polymerase reinitiate transcription with another polymerase Newly synthesised RNA is Roger Kornberg 2006 Nobel Prize for Chemistry for work on separated from the DNA structure and function of eukaryote RNA polymerase Runs through another DNA double helix enters through a groove and unwinds groove before exiting from the polymerase RNA nucleotides added to growing 3’ end of RNA molecule As it funnels through the polymerase, the DNA-RNA hybrid hits a “wall” of amino acids 21 22 Termination Pre-mRNA processing Different termination mechanisms for the 3 RNA polymerases RNA polymerase II transcribes well past the coding sequence Extra nucleotide (methylguanosine) and other of most genes (‘00s or ‘000s of nucleotides) methyl groups; only mRNA from RNA polymerase II The end of the pre-mRNA is cleaved at a specific site while 50-200 adenine nucleotides; not encoded in DNA but transcription still taking place at 3’ end of molecule added after transcription Takes place 11-30 One piece of mRNA eventually encodes the protein nucleotides downstream of a consensus sequence AAUAAA The other has 5’ end trailing from the polymerase An endonuclease attaches to this and moves towards the polymerase degrading the RNA as it goes Transcription takes place in the nucleus; translation in the cytoplasm This allows mRNA to be modified before translation Transcription is terminated when the polymerase is reached 23 24 4 RNA splicing RNA splicing Some introns have an active site and splice themselves out Most have specific sequences at 5’ and 3’ ends which are recognised by small nuclear ribonucleoproteins (snRNPs) snRNPs: complexes of proteins and specific RNA molecules Spliceosome: combination of pre-mRNA and the snRNPs Responsible for folding the pre-mRNA into correct configuration for cutting out introns and splicing (covalently) the neighbouring exons together Exon-junction complex (EJC): after splicing, proteins deposited ~20 nucleotides upstream of exon-exon junctions Pierce, 2014 which promotes export of mRNA into the cytoplasm 25 26 RNA splicing & RNA splice sites the spliceosome Majority of human introns start with GU (GT in DNA sense Interactions between strand) which is called the donor splice site and end with AG mRNA and snRNAs and which is the acceptor splice site between different snRNAs Many other GU and AG dinucleotides within exons & introns Complementary base pairings between different RNA molecules To be recognised as a splice site has to be within a broader consensus sequence enabling the binding of snRNPs Bring essential components together to Loosely defined which makes prediction from analyses of allow splicing to occur DNA or RNA sequences difficult Two transesterification However, sequence variants affecting efficiency of splice reactions to join the exons and release the lariat sites are a major cause of disease 27 28 Eukaryote gene structure Alternative splicing Alternative splicing occurs in most human genes Allows different combinations of exons to be retained in the mRNA and greatly amplifies the number of proteins that can be produced from ~20,000 protein coding genes 29 30 5 Summary of transcription The genetic code Amino acids encoded by 64 base triplets called codons The code is degenerate meaning that most of the 20 amino acids are encoded by >1 codon One consequence of this is that if helps to minimise effects (a) DNA double helix unwinds locally to allow RNA polymerase to of mutations assemble the primary transcript using the template strand and rewinds after the polymerase has moved on along the chain Codons that specify the same amino acid are synonymous and differ at 3rd base known as the “wobble” position Sequence is complementary to the template strand and identical to the sense strand Initiation codon: AUG which encodes methionine (b) The primary transcript is processed by cutting out introns and splicing exons together to form mature messenger RNA (mRNA) Stop codons: UAG, UGA, and UAA specify the end of translation 31 32 The genetic code The genetic code Reading frames: three possible sets of codons can be read Each triplet nucleotide sequence from any sequence depending on which base is chosen to or codon refers to the mRNA start. Each set is known as a reading frame sequence (not DNA) One letter symbols for the amino The initiation codon determines the reading frame of a acids also given as well as the protein coding sequence standard three letter abbreviation An open reading frame is a sequence of codons bounded by start and stop codons Universality of the genetic code: applies to all organisms Some exceptions in standard codon usage (mostly associated with start and stop codons) in mitochondrial genomes and in some unicellular organisms 33 34 Amino acids Ribosomes The R group attached to the The mRNA sequence is converted into the amino acid backbone differs in each of the sequence of a polypeptide by interaction with ribosomal 20 amino acids RNA (rRNA) and transfer RNA (tRNA) During translation, amino acids are linked together by covalent Ribosomes: organelles & the site of polypeptide synthesis bonds forming between amino group of one and carboxyl Composed of two subunits, one large and one small group of a second amino acid to form a polypeptide Contain rRNA and proteins and the rRNA large subunit acts as an enzyme linking together the amino acids Free amino group at one end (N-terminus) and carboxyl group (C-terminus) at the other By convention, the amino acid sequence of a protein is written starting at the N-terminus and moving to the C-terminus 35 36 6 Ribosomal RNA Transfer RNA Two types of rRNA genes (large and small) which encode Most tRNAs are transcribed as various types or rRNA larger precursors that are then cleaved and modified to produce mature tRNAs A precursor rRNA transcript (45S) is methylated in several places, cleaved into several intermediates, and processed Have additional bases as well as into mature rRNA molecules 4 standard ones tRNA molecules each have two Small nucleolar RNAs (snoRNAs) involved in processing attachment sites: Assembled into mature ribosomes A binding site for a specific type of amino acid A 3-base segment called an anticodon that recognises a specific base sequence in mRNA 37 38 Translation Translation - initiation Translation also has three steps: initiation, elongation, and termination The poly(A) tail of mRNA is involved in Coding sequence starts some way downstream of the 5’ end of mRNA initiation of translation The initiator AUG is embedded in a consensus sequence (the Kozak sequence). The parts of the mRNA between the 5’ end and the start codon are called the 5’ untranslated sequence (5’UT) Initiation: the 5’ cap has a critical role mRNA binds to a small ribosomal subunit, the anticodon of initiator tRNA carrying methionine binds with AUG codon of mRNA, and a large ribosomal subunit binds to the complex 39 40 Translation - initiation Translation - elongation Three sites on the Elongation: amino acids added to growing polypeptide chain moving in ribosome can be 5’ → 3’ direction occupied by tRNAs A second tRNA carrying the second amino acid pairs with the second 1. Aminoacyl or A site mRNA codon and the rRNA of the larger subunit acts as an enzyme to 2. Peptidyl or P site form peptide bond between the two amino acids 3. Exit or E site The first tRNA is released and the ribosome moves to the next codon 41 42 7 Translation - elongation Translation - elongation EF = Elongation Factor 43 44 Translation - elongation Translation 45 46 Translation Translation - termination Termination: the ribosome works it’s way along the mRNA until it meets a stop codon (UAG, UAA, or UGA) Release factors bind to the termination codon The polypeptide is released from the last tRNA The tRNA and the mRNA are both released from the ribosome The two ribosomal subunits separate completing translation The parts of the mRNA downstream of the stop signal comprise the 3’ untranslated sequence (3’UT) These 5’ and 3’ untranslated sequences are important in regulating the stability of the mRNA 47 48 8 Translation - termination mRNA surveillance mRNA surveillance: cell has several different mechanisms to deal with errors in mRNAs and to ensure accuracy of information transfer As an example, consider where a mutation alters a codon specifying an amino acid changing it to a termination codon (a nonsense mutation) Results in a premature end to translation so that resulting protein is truncated and often non-functional Nonsense mediated mRNA decay (NMD) is a process which results in rapid elimination of mRNA containing premature termination codons Mechanism for NMD appears to involve exon-junction proteins 49 50 Gene Post-translational modification regulation After translation, several further processes are needed to convert the polypeptide into a fully functional protein modifications Sometimes large precursor proteins are synthesised which must be Regulation of gene cleaved and trimmed to become functional proteins expression can occur at several stages of the The N-terminal methionine residue may be removed process of going from: Some proteins require attachment of carbohydrates for activation DNA → RNA → protein Amino acids within a protein maybe modified by – phosphates, carboxyl groups or methyl groups may be added Amino acid end of a protein often acetylated after translation Ubiquitin may be added which targets the protein for degradation The signal sequence (15 – 30 amino acids which direct proteins to a specific location) may be removed 51 52 References References Cummings (2016) Human heredity: principles and issues, 11th ed., chapter 9 Amaral et al (2008) The eukaryotic genome as an RNA machine. Science, 319, 1787-1789 Gibson (2015) A primer of human genetics, chapter 2 Baek et al (2008) The impact of microRNAs on protein output. Nature, Griffiths et al (2020) Introduction to genetic analysis, 12th ed., chapters 8 & 9 455, 64-71 Hartl & Ruvolo (2019) Genetics: analysis of genes and genomes, 9th ed., chapter 12 Bonasio et al (2010) Molecular signals of epigenetic states. Science, 330, 612-616 Klug et al (2020) Concepts of genetics, 12th ed., chapter 13 & 14 Pierce (2020) Genetics: a conceptual approach, 7th ed., chapter 13, 14 &15 Chen & Riggs (2011) DNA methylation and demethylation in mammals. Journal of Biological Chemistry, 286, 18347-18353 Strachan & Lucassen (2023) Genetics and genomics in medicine, 2nd ed., chapter 2 Clamp M et al (2007) Distinguishing protein-coding and noncoding genes Strachan & Read (2019) Human molecular genetics, 5th ed., chapters 1 & 9 in the human genome. PNAS USA, 104, 19428-19433 Djebali et al (2012) Landscape of transcription in human cells. Nature, 489, 101-108 53 54 9 References References Gingeras (2007) Origin of phenotypes: genes and transcripts. Genome Pasquinelli (2012) MicroRNAs and their targets: recognition, regulation, Research, 17, 682-690 and an emerging reciprocal relationship. Nature Reviews Genetics, 13, 271-282 Kim et al (2008) Alternative splicing: current perspectives. BioEssays, 30, 38-47 Perez-Ortin et al (2013) Eukaryotic mRNA decay: methodologies, pathways, and links to other stages of gene expression. Journal of Magistri et al (2012) Regulation of chromatin structure by long noncoding Molecular Biology, 425, 3750-3775 RNAs: focus on natural antisense transcripts. Trends in Genetics, 28, 389- 396 Ponting & Hardison (2011) What fraction of the human genome is functional? Genome Research, 21, 1769-1776 Memczak et al (2013) Circular RNAs are a large class of animal RNAs with regulatory potency. Nature, 495, 333-338 Shlyueva et al (2014) Transcriptional enhancers: from properties to genome-wide predictions. Nature Reviews Genetics, 15, 272-286 Olovnikov et al (2012) Small RNA in the nucleus: the RNA-chromatin ping-pong. Current Opinion in Genetics & Development, 22, 164-171 Spitz & Furlong (2012) Transcription factors: from enhancer binding to developmental control. Nature Reviews Genetics, 13, 613-626 55 56 The Henry Stewart Talks Biomedical & Life Sciences Collection Go to Electronic Library → Find Databases → B → Biomedical and Life Sciences Collection 2,000+ lectures by leading world experts Available at https://hstalks.com/biosci/ Eisenberg, JC (2014) Heterochromatin, epigenetics and gene expression Gibbons, R (2014) Chromatin genes and disease Dean, A (2007) The beta-globin locus 57 10

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