Gene Expression In Eukaryotes PDF

 2 PART II-C Gene expression in eukaryotes  3 Eukaryotic gene expression 4  5 GENE EXPRESSION Prokaryotes...

 2 PART II-C Gene expression in eukaryotes  3 Eukaryotic gene expression 4  5 GENE EXPRESSION Prokaryotes Eukaryotes Transcription Cytoplasm Nucleus 3 nucleus RNAP 1 1 mitochondrion 2-3 chloroplast Translation Cytoplasm Cytoplasm mRNA Often polycistronic Monocistronic 5’ UTR / 3’ UTR Shorter than eukaryotic Longer than prokaryotic (leader/trailer) mRNA mRNA mRNA processing No Yes Introns No Most genes Few proteins, few Complex regulatory Regulation regulatory sequences networks and TADs  6 Structure of Eukaryotic Gene Core promoter: ~100 bp, contains TSS: TATA box, CAAT box, GC box. Bind general transcription factors = low transcription Proximal & Distal promoters: Up to several Kbp away: Enhancers bind tissue specific transcription factors = high expression Insulators bind insulator proteins to set TAD boundaries. Silencers bind repressor proteins to inhibit transcription.  7 More on eukaryotic promoters Active promoters are in nucleosome-free zones. Flanking nucleosomes can have active or repressed epigenetic marks. During early development, promoters are bivalent/poised and can become active or inactive as the cell differentiates. Mammalian genes often have two or more alternate promoters. Respond to different regulatory elements Allow production of protein isoforms In the human genome, 70% of promoters are associated with CpG islands and lack most core promoter elements.  8 Enhancers are recognized and bound by tissue-specific transcription factors 9  10 Structure of Eukaryotic Gene RNA coding region: Contains one or more introns. Starts with an exon that contains the start codon. Ends with an exon that contains the stop codon. RNA transcripts must be post-transcriptionally processed. No clear transcription termination sequence: Determined during RNA processing. Cleavage occurs 15-30 nt downstream of PAS  mRNA Processing Protects mRNA from exonucleases. Aids mRNA transport to cytoplasm and binding to 40S. Aids mRNA transport to cytoplasm and binding to ribosome.  12 Eukaryotic Transcription: Termination CPSF: cleavage and polyadenylation specificity factor PAP: PolyA Polymerase  13 Alternative Splicing  14 Alternative splicing = different isoforms = different proteins 5’ 3’ 5’ 3’ 5’ 3’ 3’ 5’ 5’ 3’ 5’ 3’  15 38,016 Shades of DSCAM (Drosophila) The DSCAM gene (top) is 61.2 kb long and after transcription and splicing produces a 7.8 kb, 24 exon mRNA (middle). Exons 4, 6, 9, and 17 are encoded as arrays of mutually exclusive alternative exons. If all possible combinations of single exons 4, 6, 9, and 17 are used, the DSCAM gene produces 38,016 different mRNAs and proteins.  16 Prokaryote vs Eukaryote mRNA Aids: -Ribosomal binding Aids: -Export of mRNA to -Ribosomal binding cytoplasm -Removal of introns Prevents degradation Prevents degradation  17 Eukaryotic Translation More factors are required. Eukaryotic ribosome is larger than prokaryotic ribosome. Translation takes place in the cytoplasm. Eukaryotic translation is highly regulated: Initiation in particular Eukaryotic translation elongation and termination are quite similar to prokaryotic translation.  18 Flow of genetic information: Gene > mRNA > protein Promoter Exon 1 Intron Exon 2 Terminator dsDNA RNA coding region 5’ UTR Exon 1 Intron Exon 2 3’ UTR pre mRNA Signal for 3’ cleavage 5’ UTR Exon 1 Exon 23’ UTR mRNA AAAAAAAA Protein coding 5’Cap region PolyA Tail Start codon Stop codon  19 Nucleotide sequence of duck SAA (serum amyloid A protein) cDNA aligned with the predicted amino acid sequence. The predicted N-terminal signal peptide and polyadenylylation signal sequence are underlined. The amino acid sequence derived from duck SAA genotype A is shown complete and the six conserved nucleotide changes in type B are shown above.  20 Eukaryotic gene expression Regulation of transcription: Internal cellular signals: DNA binding factors Chromatin state Transcription machinery External/environmental signals Complex interplay Housekeeping genes will be constitutively expressed. Other genes have specific temporal-special expression.  21 ADDITIONAL SLIDES  22 Translation: Prokaryotes vs. Eukaryotes Prokaryotes Eukaryotes Characteristics of the code In the cell, translation occurs in the _____ mRNA life Presence/use of aminoacil-tRNA synthetases Characteristics of the ribosomal subunits (importance?) Attachment of small subunit during initiation Amino acid specified by the initiation codon Factors used during initiation, elongation and termination Presence of polyribosomes  23 5’Cap = 7-methylguanosine  24 Cap- and Poly(A) binding proteins help the mRNA engage the eukaryotic RBS  25 Both are dsDNA, have a promoter, RNA coding region and transcription terminator.  26 Structure of Eukaryotic Gene Transcription terminator Promoter: RNA coding region Core – basic transcription. Proxima/Distal – modulate rate of transcription. 27 28