PLNT 2530 2021 Unit 4 NUCLEAR GENE STRUCTURE

PLNT 25302022Unit 4NUCLEAR GENE STRUCTUREPlant Biotechnology by A Slater, N Scott, M FowlerAnalysis of genes and Genomes by Richard J ReecePlant Biotechnology and Genetics by C Neal Stewart JRUnless otherwise cited or referenced, all content of this presenataion is licensed under the Creative Commons LicenseAttribution Share-Alike 2.5 Canada1

Prokaryotes: Bacteria and ArcheaAll functions in a single cellular compartmentMenbrane functions on plasma membraneTranscription, translation and replication can all occur simultaneouslyfrom o225/chap04/ss3.htm2

Eukaryotes: Protists, Plants, Fungi, AnimalsCellular functions compartmentalized in membranous organellesTranscription in nucleus; translation in cytoplasmInterphase: Gene expression; Mitosis: DNA replication and cell divisionfrom o225/chap04/ss3.htm3

Eukaryotic genes1) ribosomal RNA (rRNA) - 4 ribosomal RNA genes from 2 transcripts2) transfer RNA (tRNA) - carry amino acids that are incorporated into proteinsduring translation.3) messenger RNA (mRNA) - translated into proteins4) heterogeneous nuclear RNA (hnRNA) - an umbrella term that encompasesa wide variety of non-coding RNAs that carry out diverse functions, manyinvolved in gene expression or processing of RNA.4

RNA polymerase locationProductsINucleolus28S rRNA, 18S rRNA and 5.8S rRNAIINucleusmRNAIIINucleustRNA, 5S rRNArRNA - requires transcription of the gene into RNA by RNApolymerase I (Pre-rRNA 18S, 5.8S, 28S genes) and RNApolymerase III for 5S RNA gene.Analysis of genes and genomes-Richard J Reece, 20045

Elements of a nuclear geneDistal tart Site TSS5’ UTRATGCAAT/ TATAAGGAPromoterRegion1000-3000 b-1 1ExonGTTAA/TAG/TGAAGIntron3’ altermination site600 30,000 bGenome Management in Eukaryotes (based on Figure 10.1 of Lee and Leagood)6

Gene structureTerminology:downstreamupstream-in the 3' direction-in the 5' directionRegulatory region of gene: is promoter present in the upstream region.-Can be proximal and distal regions.Proximal region responsible for the basal transcription and tissue specificityThis region consists of:i) Transcription Start Site (-1/ 1)-gene location where the firstribonucleotide of the RNA is being synthesized ( 1 first base of thetranscript).ii) TATA box (-25 to -30) in eukaryotes called because of theprominence of A and T’s. RNA polymerase and general TF’s bind toTATA and form pre-initiation complex.iii) Other sequences, 'CAAT‘ box (-70 to -80 bases), not required fortranscription but influence the level, rate, timing or tissue specificity oftranscription.7

Gene structureDistal sequences modulate gene expression (amplify or suppress)-influence developmentally and environmentally regulated genesand may determine tissue specificityEnhancers are a class of distal sequence elements act in aposition- and orientation-independent manner to elevate genetranscriptionCan be upstream (100 b -5 kb), downstream or even in an intron ofthe gene8

Gene structureregulatory elements – sequences (known or unknown) thatinfluence gene expression cis-elements - regulatory elements that are directly attached tothe promoter region and on the same strand as the coding regiontrans-acting factors - normally protein transcription factorswhich interact with cis-elements to affect the transcriptionprocess. They are trans in the sense that they are created ongenes far from the gene being affected9

Protein encoding genes - transcribed from TSS and processed intomRNAFeatures are related to:i) transcriptionii) post-transcriptional modification of the RNA transcript (in thenucleus)iii) export of mRNA to the cytoplasmiv) translation of the mRNA into a polypeptide10

Computer-generated video of Chromosomes,DNA replication, Transcription,and Translationhttps://www.youtube.com/watch?v 4PKjF7OumYo11

RNAP - RNA polymerase12

Transcription initiation: interactions of cis-elements andtrans-acting DNA binding proteinsDistal elementDNA bindingproteinAdaptorTFIIATFIIBTFIIETFIIDRNA pol IITATA boxTranscriptionBased on Fig 10.9 of Lea & Leegood- Genome Management in Eukaryotes13

RNA processing The primary transcript is not the mRNA found in the cytoplasm but aproduct called or pre mRNA– Plant primary transcripts typically 1-3 kb– Mammalian transcripts can be much larger Largest known (2,000 kb) Duchenne muscular distrophy gene withmore than 50 large introns, mRNA 18 kb– pre mRNA includes all introns– pre-mRNA is included in the broad category of heterogeneousnuclear RNA (hnRNA), which encompasses many types ofRNAs in the nucleus.Involves :5’Capping3’ polyadenylationIntron splicing14

TerminologyIntron is an intervening section of non-coding sequence whichinterrupts the coding/non-coding sequence of a gene and isremoved during processing of the gene primary transcript.TSSAUGPre mRNAExon any stretch of sequence from the primary transcript (andrelated region of gene) which reaches the cytoplasm as part of themRNA.15

Gene structureTranscription of gene with introns into pre mRNATSSExon 1Exon 2Intron 1Exon 3AATAAAIntron 25’ UTR3’ UTRAUGUAGAAUAAAPre mRNA5’ UTR - 5’ untranslated region - Upstream of translation start site3’UTR - 3’ untranslated region - Downstream of translation stop site16

Transcriptional processing of a nuclear geneTSSAATAAAPre mRNARNA polymerase II & cap 0Intron removalmRNAAAUAAAAAAA50-200capExport from nucleus to cytoplasm17

Post-translational steps of gene 'Export from nucleus to cytoplasmUGAUAAUAGAUGAAUAAATranslationaminoNH 3terminusCOO-carboxy terminusFolding to form secondary structure NH3ProteinCOO18

1. Cap additionOCH3NNH2NNNOHOOOOCH2O PO PO POHO-O-OCH2O-baseOO7 methyl guanylateOCH3RNA-occurs rapidly during transcription by addition of GTP in a 5’-5’ manner-methylation of the 2’-site of the second ribose occurs in all animals and higherplantsCap Function: Recognition by cap binding protein which couples with 40Sribosomal subunit for formation of translation pre-initiation complex19

2. Polyadenylation Cleavage of pre mRNA 10-30 bases downstream of AAUAAA signal byspecific RNA endonuclease Addition of 20-250 A ribonucleotide residues by a template-independentRNA polymerase –poly A polymeraseSignal Consensus sequenceFrequency % AnimalsPlantsAAUAA97 98 100 100 10090 100 100 87 91A9765Polyadenylation doesn’t occur in rRNA, tRNA genes BUT occurs for all plantmRNAs except for histone genesFunction of Poly A tail:i. intron processing,ii. transport of mRNA to cytoplasmiii. protection of transcript in cytoplasm from exonucleasedegradationTechnological use: Separation of mRNA from other RNA species20

3. Intron splicing Several classes of introns with different removal mechanisms Only consider introns of nuclear, protein-encoding genes (plastid andmitochondrial protein genes are similar) Plant introns typically 70-1000 b long and rich in A and U’s (mammalianintrons are larger (to 100kb) and more abundant Introns have characteristic boundary sequencesIntron-exon junction consensus sequenceexon intronintron exon--A G G U A A G U-----------Py n PY PY Pu A Py------N C A G G---Frequency % 62 77 100 100 60 74 84 5078 100 100 5521

Intron splicing GCCUACUCorrect splicing and translationAUGCAUUCAGGACAGGUAMet His Ser Gly GlnCAGGAUGGGCCUACUAsp Gly Pro ThrIncorrect splicing and translationAUGCAUUCAGGACAGGUAMet His Ser Gly GlnCAGGAUGGGCCUACUMet Gly Leu LeuCritical that introns be precisely removed or an incorrect protein is made22

Translation and post-translation elements of genesProtein processing into a biologically active formconversion of mRNA into linear series of amino acidsPost-translational modifications-Proteolytic processingtarget (signal) sequence removalprotein cleavage-modification of amino acidshydroxylation (Pro)phosphorylation (Ser, Thr, Tyr)glycosylation (Ser, Thr, Asn)acetylation (Lys)Information encoded in gene23

Targeting of proteins to a cellular destinationNuclear-encoded proteins are translated in the cytoplasm but aretargeted to an organelle in the cell based on signal sequences in theprotein.a) endoplasmic reticulumb) vacuoleinternal sequencesc) nucleusd) chloroplast15-60 N-terminal aminoe) mitochondriaacids which are cleaved offf)at the destination siteexport from the cellComputer programs exist which can predict localization site from thesequence24

SummarypromoterFlow for expression3'pre-mRNA 5'mRNA 5'PreproteinAAAAAAAAANPost-translational modification3'All the information isencoded in geneCNCCFolded protein at organelle destinationN25

4 Eukaryotic genes 1) ribosomal RNA (rRNA) - 4 ribosomal RNA genes from 2 transcripts 2) transfer RNA (tRNA) - carry amino acids that are incorporated into proteins during translation. 3) messenger RNA (mRNA) - translated into proteins 4) heterogeneous nuclear RNA (hnRNA) - an umbrella term that encompases a