RNA Synthesis and Processing

RNA Synthesis andProcessing

OverviewGenome structureMendelian geneticsLinkage and recombination

Chromosome structureShort arm: plong arm: q

A few termsA chromosome contains two (almost) identicalcopies of DNA molecules. Each copy is called achromatid and two chromatids are joined at theircentromeres.Chromosomes and regions in a chromosome arenamed. For example by notation 6p21.3, wemean• 6 : chromosome number• p : short arm (from petit in French), q for longarm• 21.3 : band 21, sub-band 3 (microscope)

Chromosomes in Cell Division

Chromosome Structure

Chromosome structure

Mendelian GeneticsMendel started genetics research before we knowchromosome and genePhenotype-- observable difference amongmembers in a population• For example: hair color, eye color, blood typeWhat controls a phenotype?• This is the question that Mendel tried toanswer• Is still the central question of modern geneticsHe used pea, a simple organism, and quantitativemethod to study phenotypes.• We call a quantitative study of biologycomputational biology now.

Gene linkageExperiments: white eyed female flies cross with wild typemales (red eye, dominant)What should we expect:• All red eyed flies in F1No! We have 50% red eyed and 50% white eyed• Why?• A further study show that all red eyed are female andall white eyed are male

Gene Linkage:Morgan’s explanation:wwX+Yw+wYFemale, red eyeMale, while eye

RecombinationF1 generation: white eyed, miniature wingfemale crossed to wild type maleF2 generation (ie. heterozygous femalecrossed to a wm male.We know that white eye gene and theminiature gene are in the samechromosomeWhat should we expect:• female: 50% wm, 50% normal• male: 50% wm, 50% normal

RecombinationWhat we have: F2 generation (ie. heterozygous femalecrossed to a wm male:• white eyes, normal wings 223• red eyes, miniature wings 247• red eyes, normal wings 395• white eyes, miniature wings 382We have some combination that does not appear inparents!

Recombinationchromtides exchange their DNAcomponents.

Modern GeneticsForward genetics: given a phenotype,how do we identify the genes thatcontribute to the phenotype?•Cancer, Parkinson's Disease,…Reverse genetics: given a gene, howcould we know what are thephenotype that the gene mightcontrol?•Diagnostics

How are Phenotypes Caused?Through a biochemical pathways:A W B X C Y D ZEGenes, mRNA, and proteins

Chromosome structure

Chromosome StructureChromosomes are made up of a single continuous DNA moleculecan’t be straight-- would be many times length of the cellChromatin: ordered aggregate of DNAand proteins visible in the cell cycle

Chromosome Structureheterochromatin: dense, compact structure during interphasegenerally near the centromere and telomeres (chromosome ends)composed of long tracks of fairly short base pair repeatsfew genes compared to euchromatineuchromatin: less dense DNA that only becomes visible after condensingtypically has genes being actively transcribed

Gene Expression• genes are located at specific places on thechromosome (loci)• regions corresponding to genes aretranscribed• sequences flanking the coding sequencecontrol the start and stop of transcription• not all genes are expressed at the samerates or at the same times}

RNA Synthesis and Processing• Transcription in Prokaryotes• Eukaryotic RNA Polymerases andGeneral Transcription Factors• Regulation of Transcription inEukaryotes• RNA Processing and Turnover

IntroductionRegulation of gene expression allowscells to adapt to environmentalchanges and is responsible for thedistinct activities of the differentiatedcell types that make up complexorganisms.

Transcription in ProkaryotesRNA polymerase catalyzespolymerization of ribonucleoside 5′-triphosphates (NTPs) as directed by aDNA template, always in the 5′ to 3′direction.Transcription initiates de novo (nopreformed primer required) at specificsites—this is a major step at whichregulation of transcription occurs.

Figure 7.5 Structure of bacterial RNA polymerase

Figure 7.1 E. coli RNA polymerase

Figure 7.2 Sequences of E. coli promoters

Figure 7.4 Transcription by E. coli RNA polymerase (Part 1)

Figure 7.4 Transcription by E. coli RNA polymerase (Part 2)

Transcription in ProkaryotesTranscription of the GC-rich invertedrepeat results in a segment of RNA thatcan form a stable stemloop structure.This disrupts its association with theDNA template and terminatestranscription.

Figure 7.6 Transcription termination

Figure 7.8 Negative control of the lac operon

Transcription in ProkaryotesCis-acting control elements only affectthe expression of linked genes on thesame DNA molecule (e.g. the operator).Trans-acting factors can affectexpression of genes located on otherchromosomes (e.g. the repressor).The lac operon is an example of negativecontrol—binding of the repressor blockstranscription.

Transcription in ProkaryotesAn example of positive control in E. coli :Presence of glucose (the preferredenergy source) represses expressionof genes for enzymes that breakdown other sugars, such as the lacoperon.

Transcription in ProkaryotesLow glucose levels activate adenylylcyclase, which converts ATP to cAMP.cAMP then binds to catabolite activatorprotein (CAP).CAP then binds to its target DNAsequences, 60 bases upstream of thetranscription start site in the lac operon.

Figure 7.9 Positive control of the lac operon by glucose

Eukaryotic RNA Polymerases and General Transcription FactorsEukaryotic cells have three nuclear RNApolymerases that transcribe differentclasses of genes.They are complex enzymes, consistingof 12 to 17 different subunits each.They all have 9 conserved subunits, 5 ofwhich are related to subunits ofbacterial RNA polymerase.

Figure 7.10 Structure of yeast RNA polymerase II

Eukaryotic RNA Polymerases and General Transcription FactorsPromoters contain several differentregulatory sequence elements.Promoters of different genes containdifferent combinations of promoterelements, which appear to functiontogether to bind general transcriptionfactors.

Figure 7.13 RNA polymerase II/Mediator complexes and transcription initiation

Figure 7.14 The ribosomal RNA gene

Regulation of Transcription in EukaryotesSome regulatory sequences are fartheraway—called enhancers.They were first identified during studiesof the promoter of another virus, SV40.

Figure 7.19 The SV40 enhancer

Regulation of Transcription in EukaryotesActivity of enhancers doesn’t depend oneither their distance from, or orientationwith respect to, the transcriptioninitiation site.

Figure 7.20 Action of enhancers (Part 1)

Figure 7.20 Action of enhancers (Part 2)

Figure 7.21 DNA looping

Regulation of Transcription in EukaryotesChromatin immunoprecipitationidentifies DNA regions that bind totranscription factors.Cells are treated with formaldehyde tocross-link transcription factors to the DNAsequences to which they were bound.Chromatin is extracted and fragmented.Fragments of DNA linked to atranscription factor can then be isolatedby immunoprecipitation.

Figure 7.25 Chromatin immunoprecipitation (Part 1)

Figure 7.25 Chromatin immunoprecipitation (Part 2)

Regulation of Transcription in EukaryotesOne of the first transcription factors to beisolated was Sp1, in studies of virusSV40 DNA, by Tjian and colleagues.Sp1 binds to GC boxes in the SV40promoter. This established the action ofSp1 and also suggested a method forpurification of transcription factors.

Key Experiment 7.1 Isolation of a Eukaryotic Transcription Factor: Purification of Sp1

Figure 7.28 Examples of DNA-binding domains (Part 1)

Figure 7.28 Examples of DNA-binding domains (Part 2)

Regulation of Transcription in EukaryotesThe most common is the zinc fingerdomain, which binds zinc ions andfolds into loops (“fingers”) that bindDNA.Steroid hormone receptors containzinc fingers; they regulate genetranscription in response to hormonessuch as estrogen and testosterone.

Regulation of Transcription in EukaryotesHelix-turn-helix domain: one helixmakes most of the contacts with DNA,the other helices lie across the complexto stabilize the interaction.They include homeodomain proteins,important in the regulation of geneexpression during embryonicdevelopment.

Regulation of Transcription in EukaryotesHomeodomain proteins were firstdiscovered as developmental mutantsin Drosophila.They result in development of flies inwhich one body part is transformed intoanother.In Antennapedia, legs rather thanantennae grow from the head.

Figure 7.29 The Antennapedia mutation

Regulation of Transcription in EukaryotesLeucine zipper and helix-loop-helixproteins contain DNA-binding domainsformed by dimerization of twopolypeptide chains.Different members of each family candimerize with one another—combinations can form an expandedarray of factors.

Regulation of Transcription in EukaryotesThe activation domains of transcriptionfactors are not as well characterized astheir DNA-binding domains.Activation domains stimulatetranscription by two mechanisms:• Interact with Mediator proteins andgeneral transcription factors• Interact with coactivators to modifychromatin structure.

Figure 7.30 Action of transcriptional activators

Regulation of Transcription in EukaryotesGene expression in eukaryotic cells isalso regulated by repressors whichbind to specific DNA sequences andinhibit transcription.In some cases, they simply interfere withbinding of other transcription factors.Other repressors compete withactivators for binding to specificregulatory sequences.

Figure 7.33 Decondensed chromosome regions in Drosophila

Regulation of Transcription in EukaryotesHistone modification provides amechanism for epigenetic inheritance—transmission of information that is notin the DNA sequence.Modified histones are transferred to bothprogeny chromosomes where they directsimilar modification of new histones—maintaining characteristic patterns ofhistone modification.

Figure 7.36 Epigenetic inheritance of histone modifications (Part 1)

Figure 7.36 Epigenetic inheritance of histone modifications (Part 2)

Regulation of Transcription in EukaryotesChromatin remodeling factors areprotein complexes that alter contactsbetween DNA and histones.They can reposition nucleosomes,change the conformation ofnucleosomes, or eject nucleosomesfrom the DNA.

Figure 7.37 Chromatin remodeling factors

Regulation of Transcription in EukaryotesTranscription can also be regulated bynoncoding RNA molecules, includingsmall-interfering RNAs (siRNAs) andmicroRNAs (miRNAs).They can induce histone modificationsthat lead to chromatin condensationand formation of heterochromatin.

Regulation of Transcription in EukaryotesIn the yeast S. pombe, siRNAs directformation of heterochromatin atcentromeres, by associating with theRNA-induced transcriptional silencing(RITS) complex.RITS includes proteins that inducechromatin condensation andmethylation of histone H3 lysine-9.

Figure 7.38 Regulation of transcription by siRNAs

Regulation of Transcription in EukaryotesDNA methylation is another generalmechanism that controls transcriptionin eukaryotes.Methyl groups are added at the 5-carbonposition of cytosines (C) that precedeguanines (G) (CpG dinucleotides).

Figure 7.40 DNA methylation

Regulation of Transcription in EukaryotesMethylation is common in transposableelements, it plays a key role insuppressing the movement oftransposons.DNA methylation is associated withtranscriptional repression of somegenes, and also has a role in Xchromosome inactivation.

Regulation of Transcription in EukaryotesDNA methylation is a mechanism ofepigenetic inheritance.Following DNA replication, an enzymemethylates CpG sequences of adaughter strand that is hydrogenbondedto a methylated parentalstrand.

Figure 7.41 Maintenance of methylation patterns

Regulation of Transcription in EukaryotesDNA methylation also plays a role ingenomic imprinting: expressiondepends on whether they are inheritedfrom the mother or from the father.Example: gene H19 is transcribed onlyfrom the maternal copy. It is specificallymethylated during the development ofmale, but not female, germ cells.

Figure 7.42 Genomic imprinting

RNA Processing and TurnoverMost newly-synthesized RNAs must bemodified, except bacterial RNAs whichare used immediately for proteinsynthesis while still being transcribed.rRNAs and tRNAs must be processed inboth prokaryotic and eukaryotic cells.Regulation of processing steps providesanother level of control of geneexpression.

RNA Processing and TurnoverThe ribosomal RNAs of both prokaryotesand eukaryotes are derived from asingle long pre-rRNA molecule.5S rRNA in eukaryotes is transcribedfrom a separate gene.

Figure 7.43 Processing of ribosomal RNAs

RNA Processing and TurnovertRNAs also start as long precursormolecules (pre-tRNAs), in bothprokaryotes and eukaryotes.Processing of the 5′ end of pre-tRNAsinvolves cleavage by the enzymeRNase P.RNase P is a ribozyme—an enzyme inwhich RNA rather than protein isresponsible for catalytic activity.

RNA Processing and TurnoverRNA editing is processing (other thansplicing) that alters the protein-codingsequences of some mRNAs.It involves single base modificationreactions such as deamination ofcytosine to uridine and adenosine toinosine.

RNA Processing and TurnoverUltimately, RNAs are degraded in thecytoplasm.Intracellular levels of any RNA aredetermined by a balance betweensynthesis and degradation.Rate of degradation can thus controlgene expression.

RNA Processing and TurnoverBacterial mRNAs are rapidly degraded,most have half-lives of 2 to 3 minutes.Rapid turnover allows the cell to respondquickly to changes in its environment,such as nutrient availability.

RNA Processing and TurnoverIn eukaryotic cells, mRNA half-lives vary;less than 30 minutes to 20 hours inmammalian cells.Short-lived mRNAs code for regulatoryproteins, levels of which can vary rapidlyin response to environmental stimuli.mRNAs encoding structural proteins orcentral metabolic enzymes have longhalf-lives.

RNA Processing and TurnoverDegradation of eukaryote mRNAs isinitiated by shortening of the poly-Atails.Rapidly degraded mRNAs often containspecific AU-rich sequences near the 3′ends which are binding sites forproteins that can either stabilize themor target them for degradation.

Figure 7.56 mRNA degradation

RNA Processing and TurnoverThese RNA-binding proteins areregulated by extracellular signals, suchas growth factors and hormones.Degradation of some mRNAs isregulated by both siRNAs and miRNA.