YR1 Lecture - Genetics 2 Gene Expression and Regulation 2024 PDF

COPYRIGHT COMMONWEALTH OF AUSTRALIA Copyright Regula=ons 1969 WARNING This material has been reproduced and communicated to you by or on behalf of University of Western Sydney pursuant to Part VB of the Copyright Act 1968 (the Act). The material in this communicaDon may be subject to copyright under...

COPYRIGHT COMMONWEALTH OF AUSTRALIA Copyright Regula=ons 1969 WARNING This material has been reproduced and communicated to you by or on behalf of University of Western Sydney pursuant to Part VB of the Copyright Act 1968 (the Act). The material in this communicaDon may be subject to copyright under the Act. Any further reproducDon or communicaDon of this material by you may be the subject of copyright protecDon under the Act. Do not remove this noDce. 1 The central ↑ immature DNA TRANSCRIPTION > - & revesse Replicatioe NSCRIPTION RNA 'cytoplasm nucleus dogma huRNACheterogenous posttranscription > ↓ I - splicing translation mRNA a "Protein MATURE synthes - amino acids (messager of INTRONS - BAD GUS 2 5 cawil I Types nucleolus ② rRNA (abundant) ② mRNA "codom" ③ tRNA "anti-codon" > - RNA polymerase I nucleus > - RNA polymerace # nucks > - RNA polymerase # PROTEINS Genetics 2 Gene expression and regulation Dr. Elizabeth O’Connor [email protected] Learning objectives Describe the processes and enzymes involved in transcription and translation of DNA Explain how proteins are made using knowledge about DNA transcription and translation and amino acid structure 3 Lecture overview Genes and gene expression Central dogma of molecular biology Transcription Post-transcription modifications Translation Forming functional proteins Quick intro to mutagens 4 Structure of coding material What is DNA? Nuclear material containing the instructions to build a living organism Made up of nucleotide base pairs (A, T, G, C) on a sugar backbone What is a gene? Sequence of nucleotide basepairs (bp) that code for a protein The hereditary unit, passed from parent to offspring, one copy/gene from each parent 6 Gene expression The process of using the gene information/code to produce a functional product Functional products = proteins, functional non-coding RNAs ~ functional Tightly controlled spatially and temporally (developmentally) space & time & 7 What is RNA? DNA - A Ribonucleic acid Sugar backbone is Ribose rather than Deoxyribose Single stranded, tends to be much shorter than DNA Made from the nucleotide bases C, G, A and U (instead of T) 8 Klug and Cummings (1997) Types of RNAs mRNA (messenger RNA) – involved in protein production form to out -> By translating the code takes & - Produced by RNA polymerase II the DNA forms Later a poster tRNA (transfer RNA) – involved in protein production Stage - Produced by RNA polymerase III rRNA (ribosomal RNA) – structural component of ribosome - Produced by RNA polymerase I and III Other ncRNAs (non-coding RNAs) - Produced by RNA polymerase I, II and III 9 Non-coding RNAs Do not produce funcLonal proteins as end products Many and varying roles Nc = non-coding Nt = nucleotide 10 Central dogma of molecular biology 5’ Exon 1 DNA Intron Exon 2 Intron Exon 3 3’ Transcription Pre-mRNA mRNA Processing Mature mRNA AAAAAAAAAAA Translation Translated Region Untranslated Region Protein 11 Gene structure Gene structure includes protein coding sequences and regulatory sequences Promotor = binding site for RNA pol II Exon = Expressing region (exit the nucleus) Intron = Intragenic/intervening region (removed from final mRNA, remain inside nucleus) Poly A site = region where repeated A nucleotides are added in mRNA processing Start/stop codon = the sequence of nucleotides that code for the start and stop of amino acid chain production (translation) GENE Start of transcription Core promoter Upstream regulatory elements RNA produced starting point of Exon 1 transcription DNA ↓ where polymer RNA magnes binds ass start its work to Exon 3 Exon 2 Intron 1 Poly A site Intron 2 Translated region Start codon ATG Stop codon 12 So what do the “other” bits do? Introns – expression regulaLon -> ncRNAs, alternaLve splicing, nuclear export 5’ UTR – ribosome recogniLon sequence for translaLon 3’ UTR – translaLon terminaLon, expression regulaLon Poly A tail – nuclear export, translaLon, mRNA stability Promoter and regulatory sequences – bind nuclear proteins to regulate transcripLon Upstream regulatory elements Core promoter Start of transcription RNA produced Exon 1 Intron 1 5’ UTR Start codon ATG Exon 3 Exon 2 DNA Poly A site Intron 2 Translated region 3’ UTR Stop codon 13 Codons The language of genetics is written with the nucleotide letters C, G, A, T/U The letters are arranged into words called codons Codons are sequences of 3 nucleotides Each set of 3 nucleotides codes for a different amino acid One codon is for START (Methionine) (Note: this is also a stand alone amino acid) The gene reading frame starts at START Three codons are for STOP (Amber, Ochre, Opal/Umber) (Note: these are not amino acid names) - 14 Transcription Transcription The reproduction of the genetic code as an intermediary RNA product =mRNA (messenger RNA) Produced by enzyme RNA polymerase II 4 steps – initiation, elongation, termination, processing (Mitosis/meiosis) (-> RNA -> protein) 16 Transcription - initiation Requires unwinding of DNA One of the unwound strands is the template strand – used to produce the mRNA The other strand is the coding strand – the sequence of amino acids can be read from here TranscripLon start site is designated +1 TATA box sequence defines promoter binding site TFs = transcripLon factor proteins that form the transcripLon complex with RNA pol II 17 Transcription - elongation "build" RNA is built 5’ to 3’ along the template strand All sequences within the gene (introns and exons) are transcribed Wherever RNA pol II encounters an A, it pairs a U (uracil) rather than a T (thymine) 18 Transcription - termination RNA pol II just keeps on transcribing… Set of 2 sequences signals binding of termination complex Complex cleaves pre mRNA off Complex adds Poly A tail An exonuclease digests un-needed RNA Once it catches RNA pol II, it releases RNA pol II from DNA strand I * Theories on termination – process not yet completely understood 19 Transcription video Video from the NDSU virtual cell series – oversimplified view of transcription, but good for basics (2:30) https://www.youtube.com/watch?v=WsofH466lqk 20 DNA huRNA - ↑W Mature = mRNA I Post Transcription M Post-transcriptional processing Once transcription is complete, the mRNA must be processed 3 main events – 5’ capping, 3’ polyadenylation, splicing Within - the nucens All occur in the nucleus prior to export ↑ adenocyte t to m degration. 22 Capping and tailing 5’ Cap 5’ capping involves 3 enzymes adding a methylated guanosine residue The cap protects the RNA from degradation whilst in transport to the ribosome Involved in ability of mRNA to export nucleus Recruitment of ribosomes for translation uses recognition of 5’ cap 3’ Poly A tail Termination complex contains factors that add ~200 adenosine residues = Poly A tail Also involved in mRNA protection, nuclear export and translation Length of PolyA tail determines mRNA half life 23 Splicing Removal of introns ↳ - Ability to join exons together in varying arrangements -> gene isoforms -> >10x increase in the number of transcripts that can be produced Alternate transcripts produce different proteins – different roles/locations pre processed ↓ Q ③ ② ↑ all different proteive different > - - > - 24 Other types of RNA processing MicroRNAs (miRNA) and small interfering RNAs (siRNA) (among others!) L L RNAi (interference) pathways C Small RNA sequences that bind to ss mRNA DsRNA is degraded by nucleases Interference I is where still we produce have , an but the T single MRNA RNA can not be translated and degraded gets L on gointo stranded. to o protein 25 Transcription ↓ in the occus nudlan Translation Travelation inMe occurs cytoplasm. Translation Use of the mRNA sequence to form an amino acid chain Requires mRNA and translaOonal machinery Every codon (3nt) codes for an amino acid Amino acids joined together -> protein 27 The genetic code a Phenylketonuria ↑ of in ts e important or / / the body precessor - for serotoin NIT star No – you don’t need to memorise this 28 Translational machinery - tRNA (Loops) Hairpin-like structure with an amino acid attached at the 3’ end Triplet nucleotide sequence at the base = anticodon Anticodon is complementary to the mRNA codon sequence tRNA genes transcribed by RNA pol III 29 Translational machinery - ribosome Combination of ribosomal RNA (rRNA) (2/3) and proteins (1/3) The large (60S) and small (40S) subunits come together for translation 3 slots for tRNA-binding: A, P, E 1 slot for mRNA-binding Sprobio 60S subunit - 40S subunit -° - 30 Translation - initiation tRNA with the START codon binds to 40S ribosome subunit (via binding an initiation factor eIF-3) This structure is guided to the 5’ mRNA cap and scans along the mRNA until it matches the tRNA anticodon (UAC) with the corresponding mRNA codon (AUG) The 60S ribosome subunit then binds so that the START tRNA is positioned at the P site Khan academy 31 Translation - elongation Peptide bond joins amino acid at P site (N terminus) to the one at A site (C terminus) via peptidyl transferase enzyme Next tRNA with matching anticodon arrives at A site mRNA is pulled across one codon; empty tRNA move to E site and leaves; growing chain tRNA moves to P site; A site is now empty tRNA at P site is empty 32 Khan academy Translation - termination Release factor binds to A site – carries no amino acid Tina P site enzyme (peptidyl transferase) hydrolyses bond between final amino acid and its’ tRNA Release factor is ejected; ribosome subunits disband; mRNA is released 33 Pearson Education Ltd Translation video Good video from the NDSU virtual cell series (3:15) https://www.youtube.com/watch?v=5bLEDd-PSTQ 34 Post Translation Post-translational modifications Can occur at any stage of protein life, but many are modified directly posttranslaLon Designed to mediate funcLonal protein folding or cellular localisaLon Usually mediated by enzymes Most common modificaLon is phosphorylaLon 36 Making a protein Now we have an amino acid chain…but we need a protein Primary Secondary Tertiary Quaternary Lumen Learning 37 Primary and secondary folding The primary structure is the chain of amino acids joined by peptide bonds A denatured (eg. heated) protein assumes primary structure Secondary structures include α–helices and β–pleated sheets – held together by hydrogen bonds Lumen Learning 38 Tertiary and quaternary folding Tertiary structure is the final 3D shape of the protein made from an amino acid chain Determined by various bonds/interactions Protein may be functional at this stage Quaternary structure is the assembly of protein subunits (different amino acid chains) into into a funcLonal protein Not all proteins require this level of folding This eg. is hemoglobin (2α and 2β polypepLdes with 4 heme groups) 39 Mutations affect expression Causes of mutations Mutagens in -changes and – Radiation (eg. UV) the DNA sequence how a DNA sequence they affectbe read and used can by the. body – ROS (reactive oxygen species) – Deaminating agents, alkylating agents, aromatic amines – Base analogs, intercalating agents – Biological agents (viruses/bacteria) Environmental mutagens UV and Ionizing Radiation Direct DNA damage Indirect production of free radicals Cigarette smoke >60 confirmed carcinogens DNA adducts (DNA bound to chemical) Chemotherapeutic drugs Variety of genotoxic effects Potential to increase risk of secondary mutations and drug resistance Szikriszt et al., 2016; A comprehensive survey of the mutagenic impact of common cancer cytotoxics; Genome Biol. 17:99. UV and Ionizing radiation (IR) Sun; X-ray/CT/radiotherapy Direct DNA damage -> pyrimidine dimers -> single stranded breaks Free radical damage – oxidized G pairs with A (-> G-T transversions) Why is this a problem? -> Transcriptional machinery can’t “read” through dimers or ss breaks 43 Summary DNA and gene revision RNA structure and types – mRNA, tRNA, rRNA, ncRNAs Central dogma of molecular biology: DNA -> RNA -> protein The structure and language of a gene – components and codons TranscripTon to produce an mRNA - iniTaTon, elongaTon, terminaTon Post-transcripTonal modificaTons - capping, tailing, splicing, RNAi TranslaTon – the geneTc code and translaTonal machinery - iniTaTon, elongaTon, terminaTon Post-translaTonal modificaTons Protein folding to form a funcTonal protein - secondary, terTary, quaternary UV/ionising radiaTon cause mutaTons by direct and indirect mechanisms 44 Thank you! Please provide anonymous lecture feedback via this short Qualtrics survey