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Transcription Translation ER I Eukaryotic Gene Structure a Genetic code i Codons of 3 nucleotides encode each AA ii Some AA are encoded by more than one codon redundancy iii In mRNA the code for the protein starts at AUG Met START codon and ends at one of three possible STOP codons UAA UAG UGA iv The START and STOP codons are not the first and last 3 bases of the mRNA b Control region regulatory DNA sequence i On 5 end ii Determine when gene turned on off c Exons protein coding sequences and introns enable differential splicing of exons to encode different protein isoforms d Transcription factor proteins bind to specific DNA sequences in gene control region regulate expression transcription of transcription unit e Transcription yields primary mRNA transcript of gene II Transcription i Entire exon intron sequence a Gene DNA code transcribed to form primary mRNA message b Accomplished by RNA polymerase c Occurs in nucleus of cell d Primary mRNA mature mRNA i Addition of cap to 5 end ii Addition of poly A tail to 3 end iii Splicing 1 Removal of introns 2 Splicing together of exons contain protein coding sequence 3 Differential splicing of exons generates proteins isoforms versions 4 Cell specific splicing of fibronectin mRNA generates mRNAs that encode different isoforms of the fibronectin protein a Fibronectin expressed by fibroblasts synthesize connective tissue differs from that expressed by hepatocytes liver cells Integrins 9 1 and 4 1 bind to EIIIA domain b III Translation ribosomes in cytoplasm a mRNA protein b Mature mRNA moves to cytoplasm c Ribosome Structural RNA protein complex i ii Large 60S and small 40S subunit iii RNA catalyzes formation of peptide bond b w AA to make protein d Ribosome assembles on 5 end of mRNA and scans for start AUG codon i Sets reading frame no punctuation in code continuous string ii Ribosome matches tRNA anticodon with mRNA codon e tRNA amino acid complex i tRNA carries AA linked to a specific anticodon base sequence by tRNA synthetase f Ribosome 3 sites 1 Anchors elongating peptide to i P peptide site ribosome ii A amino acid site 1 Site where incoming tRNA AA complex anticodon matches to mRNA codon iii E tRNA ejection site 1 tRNA lacking AA ejected iv shift of elongating peptide from A to P site g Addition of AA formation of peptide bond h Translation Termination i Ribosome translate terminates at one of the three stop codons ii Ribosome dissociates from mRNA and releases newly synthesized peptide i Newly synthesized protein folds to make secondary teritary and quarternary structures Some stay in cytoplasm others targets to other destinations j D N mRNA A IV Protein Targeting a Proteins encoded by nuclear genes targeted for i Cytoplasm ii Import into cytoplasmic organelles nucleus mitochondria chloroplasts ER Golgi lysosomes iii Plasma membrane iv Secretion from cells V Secretory Pathway a Secreted integral PM and lysosomal proteins b The 3 proteins have N terminal region signal sequence SS or zip code i No single motif ii 6 12 mostly hydrophobic AA cleaved from protein by signal protease in ER iii Experimental strategy proves activity of SS zip code sequence 1 Removal of SS from protein normally secreted protein remains in cytoplasm 2 Addition of SS to cytoplasmic protein protein secreted from cell c Secreted proteins i Synthesized by ribosomes associated with ER and processed in Golgi ii Yeast secretory sec mutations 1 2013 Nobel prize 2 Secretory process of humans and yeast is very similar 3 Model organism iii Exp newly synthesized protein in ER immediately after synthesis 1 Homogenize cells making protein for secretion ER fragments into resealed vesicles called microsomes 2 Experimental group Add protease to microsomes a Newly synthesized proteins inside ER microsomes protected from destruction 3 Control addition of detergent a Dissolves ER membrane iv Exp co translational insertion into ER 1 Proteins synthesized by in vitro translation system 2 Addition of microsomes to pre synthesized protein no import 3 Additions of microsomes before synthesis import and cleavage of N terminal signal sequence b Proteins degraded by added protease v Ribosome ER docking mechanism 1 SS at N terminus emerges from ribosome as protein is synthesized 2 Signal Recognition Particle SRP binds SS 3 SRP binding stops protein synthesis a Blocks A site 4 Signal Recognition Particle RNA protein complex a SRP i Structural RNA ii 6 proteins b SRP Proteins i P54 G protein 1 GDP forms binds SS 2 SS binding causes GTP exchange for GDP 3 Methionines bind SS 1 Blocks A site of ribosome prevents protein elongation ii P9 p14 iii P68 p72 1 Required for ER docking and co translational protein import 5 Interaction of SRP with SRP receptor on ER a GTPase activity of SRP p54 and SRP receptor SRP from SS removes block to protein synthesis b Docks ribosome on translocon w elongating protein in removes open translocon channel i Ribosome gated channel 6 Protein enters ER co translationally during synthesis in unfolded state 7 SS cleaves by ER signal peptidase 8 Folding of protein provides energy for translocation of protein d Integral PM proteins through channel i ER synthesis of soluble and integral membrane proteins ii Topologies of PM proteins 1 Single or multiple membrane spanning domains 2 N or C terminus on either side of membrane a Exoplasmic space can be ER Golgi lumen or cell exterior iii Type 1 PM proteins 1 One TM domain non cytosol N terminus 2 N terminal SS initiates translocation SRP SRP 3 TM domain functions as stop transfer sequence a Hydrophobic AA s 4 Ribosome dissociates from translocon a Completes protein synthesis b C terminus in cytosol 5 Stop transfer anchor sequence escapes translocon laterally into membrane bilayer iv Multiple TM domain proteins 1 N terminal SS initiates translocation SRP SRP 2 TM domain stop transfer sequence 3 Alternating TM domains function as start transfer and stop transfer sequences 4 TM domains can be predicted by hydropathy plots VI ER Post translational protein modifications maturation a GPI anchorage b Glycosylation i O linked glycosylation 1 Ser Thr linked in many proteins e g LDLR 2 Hydroxylproline linked in collagen 3 Added one sugar at a time in ER in Golgi c Formation of disulfide bonds 4 Glycosyltransferases ER resident proteins a Catalyze resident proteins ii N linked glycosylation 1 Transfer of complex from carb of Dolichol to N X T motif on protein during synthesis 2 Trimming of carb by ER resident glycosyl processing


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FSU PCB 3134 - Transcription-Translation; ER

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