Gene structure, expression and regulation in eukaryotes

Gene structure, expression and regulation in eukaryotes

1. Eukaryotic Gene Structure

   • Monocistronic - Each protein is coded for by a single separate gene
     • Advantage - Flexible and Resilient
     • Disadvantage - Complex
   • Promoter
     • Controls expression level and timing
   • Enhancers
   • Coding region
     • Nucleotides encoding peptide or RNA
     • Exon
   • poly(A) site
     • mRNA stability
       • Expression Level
   • Splice sites
     • Isoforms
   • Non coding regions
     • Introns
       • Help create genetic diversity

2. Transcription

   • Occurs in Nucleus
   • Transcription Units
     • Simple
       • 1 mRNA = 1 protein
       • Mutation along entire length will impact protein
     • Complex
       • Encodes for multiple proteins through the use of Alternative Splicing
         • Alternative in/exclusion
           • Diff composition e.g. Fibronectin
         • Alternative Promoters
           • Diff timing e.g. Pax6, Diff N termini, Diff strength of expression
         • Alternative poly(A) sites
           • Different C-termini - Heavy chain IgM
   • 3 different polymerases
   • Initiation
     • Promoter Recognition accessory proteins bind to promoter sequence promoter sequence --> recruitment of > recruitment of specific RNA polymerase
       • Accessory Proteins
         • General Transcription Factors
           • Bind to core promoter to assemble Basal Transcription Apparatus (BTA)
             • BTA includes TFs, mediator complex and RNA pol II for minimal transcription
             • Core - immediately upstream of transcription start site
         • Transcriptional Activator Proteins
           • Bind to specific DNA sequences (regulatory, promoter, enhancers, etc) to stimulate assembly of BTA
             • Leads to increased transcription
             • Allows regulation in response to different stimuli
             • Regulatory promoter is further upstream TAPs bind to DNA sequences then interacts with BTA, influences the rate of transcription initiation
               • Includes enhancer region that is even further upstream
     • TFIID binds to TATA box in core promoter
       • Then TFs and RNA pol II bind to core promoter
         • TAPs bind to sequences in enhancers
           • DNA loops out allowing the proteins bound to the enhancer to interact with BTA
             • TAP bind to sequences in the regulatory promoter and interact with BTA through the mediator
   • Elongation
     • After 30 bp of mRNA After 30 bp of mRNA synthesis Pol leaves synthesis Pol leaves promoter and TF promoter and TF behind
     • 8 nucleotides of RNA 8 nucleotides of RNA remains paired with remains paired with DNA
   • Termination
     • RNA pol II transcribes well past the coding region of many genes
       • Cleavage is near 3' end of RNA, while RNA pol II keeps transcribing
         • The RAT1 exonuclease attaches to the 5' end of the trailing RNA and moves towards the RNA pol degrading the RNA as it goes along
           • When RAT1 reaches the pol transcription is terminated

3. Translation

   • Occurs in Cytoplasm
   • Initiation
     • Small subunit binds to 5’ cap and scans mRNA for start codon Kozak sequence aids recognition
       • Initiation complex recognises 5' Cap
         • Initiation Complex - small subunit, IF, initiator tRNA (Met small subunit, IF, initiator tRNA (Met--tRNA
       • Start codon = AUG
       • requires at least 7 IF (function: separation of SU of requires at least 7 IF (function: separation of SU of Ribosome, recognition of 5’ cap, RNA helicase activity, Ribosome, recognition of 5’ cap, RNA helicase activity, recruitment Met-tRNA
       • poly(A) - bound proteins interact with 5' cap - enhances binding of small subunit at 5' of mRNA
         • Eukaryotic Ribsomes - Bigger and more complex than complex than prokaryotes but prokaryotes but same structure and same structure and function
   • Elongation
     • at least 3 elongation least 3 elongation factors equivalent to prokaryotic factors equivalent to prokaryotic EFs
       • eeF2 - translocation
   • Termination
     • Similar to prokaryotes
       • eRF1 - recognises termination codons
       • eRF2 GTP, stimulates release of polypeptide from ribosome

4. RNA processing

   • RNA molecules are processed before leaving Nucleus
   • Includes removal of parts of the primary transcript
   • Addition of 5' cap
     • 5' cap functions include - Increases the stability of the transcript •Is recognised by the ribosome to begin translation •Has a role in transport of the mRNA from the nucleus to the cytoplasm
   • RNA editing
   • RNA splicing
     • takes place in nucleus
     • order of exons in DNA is usually maintained
     • Occurs at short conserved sequences
     • Proceeds via two sequential trans-esterification reactions
     • Catalysed by a spliceosome
       • The spliceosome consists of: -5 small RNA molecules called snRNAs: U1, U2, U4, U5, U6. -Proteins.
         • Each snRNA interacts with specific proteins to form snRNPs (small nuclear riboproteins, or snurps)
     • Exceptions to normal splicing
       • Trans-splicing (e.g. nematodes and trypanosomes) joining of exons from different mRNAs
       • minor splicing: different consensus sequences and different spliceosome composition
       • Self-splicing introns (e.g. protists, mitochondria): remove themselves (no other enzyme/protein)
   • 3' cleavage and addition of poly(A) tail
     • pre-mRNA is transcribed with a consensus sequence
       • pre-mRNA is cleaved 11-30 bp upstream of consensus sequence - this cleave site contains a u rich sequence
         • poly(A) tail is added through polyadenylation
           • Functions of poly(A) tail include increasing stability and acting as a timer. Also is recognised in protein synthesis (attachment of ribosome)

5. Gene regulation

   • mRNA degradation control
     • legnth of poly(A) tail controls stability and acts as a timer
     • cleavage inside mRNA
     • 3’->5’ removal nucleotide
     • removal of 5’ CAP, 5’->3’ removal nucleotid
     • 5’UTR, CDR, 3‘UTR can affect mRNA stability
     • RNA interferecne
       • Dicer
       • RNA Induced Silencing Complex (RISC)
       • pairing with complementary sequence of mRNA -> cleavage of mRNA
       • 4 possible outcomes
         • Cleavage of mRNA
           • Double stranded RNA is cleaved by dicer enzyme to produce small interfereing RNAs (siRNAs)
             • siRNAs combine with RISC complex and pair with complementary sequences of mRNA
               • Complex cleaves mRNA and afterwards the RNA is degraded
         • Inhibition of translation
           • miRNAs produced then combine with RISC and pair imperfectly to an mRNA, which leads to inhibition of translation
         • Transcriptional silencing
           • miRNAs bind to comp sequences and attract methylating enzymes, this mehtylates the DNA or histones inhibiting (silencing) transcription
         • Degradation of mRNA
   • Translation Control
     • e.g. T cells activation mRNA already there, increase in available initiation factors for translation -> IF allow ribosomes to bind to mRNA -> TRANSLATION
   • RNA Processing Control
     • Alternative splicing
       • e.e sexual phenotype in drosophila
         • In males upstream splice site used - premature stop codon so no functional protein is produced - and in females sxl protein causes downstream splice site to be used - stop codon is spliced out alongside intron and tra protein normally produced
   • Transcriptional Control
     • Transcriptional Repressors
       • - Bind to silencers: can be some distance from regulated gene, position and orientation independent they compete with activators
       • may bind to sites near activator site -> prevents contact of activator with BTA
       • Directly interfere with assembly of BTA
     • Enhancers stimulate any promoter in vicinity
     • Insulators block effect on promoters nearby, some also limit spread of de/condensation of chromatin
     • Transcriptional Stalling
       • transcription for few bp, then pause until external stimulus is encountere
       • e.g. Heat shock in drosophila
     • Coordinated Gene Regulation - certain genes activated by same stimulus -> response element (same regulatory sequence, that provides binding sites for transcriptional activators
       • single gene can be activated by several different response elements - > activation through different stimuli
       • One stimulus can be activated by several genes --> presence of same response element
     • Combinatorial Gene Control
       • e.g. ey gene in drosophila
   • Protein activity control
     • Effects transport, function and activity
   • RNA Transport and localisation control
     • Transport mRNA across nuclear envelope
       • poly(A) and 5' cap involved

6. Epigenetics

   • changes in phenotype or gene expression by mechanisms other than changes in the underlying DNA sequence
     • may be continuous over lifespan of cell or even hereditary
   • Chromatin remodelling
     • Histone modification - A combination of diff molecules attach to tail of histone molecule. These alter the activity of the DNA surrounding them
     • Can make oncogenes more active
   • DNA methylation
     • Methyl marks added to certain bases represses gene activity
       • e.g. Gene silencing by adding methyl group to cytosine
       • Can play a role in cancer by silencing tumour suppresor genes
   • Plays a role in development - cell differentiation
     • Epigenetic reprogramming = Reproductive cells: specialised cells, lots of epigenetic tags most epigenetic tags removed in early embryo, so that cells can form every type of cell in body: “blank slate"
       • Some genes escape this reprogramming - Epigenetic inheritence