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AP Details for Protein Synthesis 2014 From gene to protein Transcription from DNA nucleic acid language to RNA nucleic acid language AP Biology Transcription • Making mRNA – transcribed DNA strand = template strand – untranscribed DNA strand = coding strand • same sequence as RNA – synthesis of complementary RNA strand • transcription bubble – Enzyme involved • RNA polymerase 5 DNA C G 3 A G T A T C T A A G C rewinding 5 A T C G T A C T 3 G C mRNA build RNA 53 G coding strand C A U C G U G T A C A G RNA polymerase T T A A C T A G C T G A T 3 5 unwinding template strand RNA polymerases • 3 RNA polymerase enzymes – RNA polymerase 1 • only transcribes rRNA genes • makes ribosomes – RNA polymerase 2 • transcribes genes into mRNA – RNA polymerase 3 • only transcribes tRNA genes – each has a specific promoter sequence it recognizes Which gene is read? • Promoter region – binding site before beginning of gene – TATA box binding site – binding site for RNA polymerase & transcription factors – Enhancer region – binding site for activators (activate genes) – Silence region – Binding site for repressors (turns genes off) What are Transcription Factors? • Initiation complex – transcription factors bind to promoter region • suite of proteins which bind to DNA • turn on or off transcription – trigger the binding of RNA polymerase to DNA Matching bases of DNA & RNA • Match RNA bases to DNA bases on one of the DNA strands A G U A G G U U C A A G C C G A U A C 5' RNA A C C polymerase A U U 3' G T G G T A C A G C T A G T C A T C G T A C C G T C Eukaryotic genes have junk! • Eukaryotic genes are not continuous – exons = the real gene • expressed / coding DNA introns come out! – introns = the junk • inbetween sequence intron = noncoding (inbetween) sequence eukaryotic DNA exon = coding (expressed) sequence mRNA splicing • Post-transcriptional processing – eukaryotic mRNA needs work after transcription – primary transcript = pre-mRNA – mRNA splicing • edit out introns – make mature mRNA transcript intron = noncoding (inbetween) sequence ~10,000 base eukaryotic DNA exon = coding (expressed) sequence pre-mRNA primary mRNA transcript mature mRNA transcript ~1,000 base spliced mRNA Splicing must be accurate • No room for mistakes! – a single base added or lost throws off the reading frame AUGCGGCTATGGGUCCGAUAAGGGCCAU AUGCGGUCCGAUAAGGGCCAU AUG|CGG|UCC|GAU|AAG|GGC|CAU Met|Arg|Ser|Asp|Lys|Gly|His AUGCGGCTATGGGUCCGAUAAGGGCCAU AUGCGGGUCCGAUAAGGGCCAU AUG|CGG|GUC|CGA|UAA|GGG|CCA|U Met|Arg|Val|Arg|STOP| RNA splicing enzymes • snRNPs – small nuclear RNA • Spliceosome snRNPs snRNA intron exon – several snRNPs 5' – recognize splice site sequence • cut & paste gene exon 3' spliceosome 5' 3' lariat No, not smurfs! “snurps” mature mRNA 5' exon 5' 3' exon 3' excised intron More post-transcriptional processing • Need to protect mRNA on its trip from nucleus to cytoplasm – enzymes in cytoplasm attack mRNA • protect the ends of the molecule • add 5 GTP cap – Chemically modified molecule of GTP – It facilitates the binding of mRNA to the ribosome and protects the mRNA from being digested by ribonucleases – enzymes in cytoplasm that break down RNA • add 3’poly-A tail – longer tail – 100-300 adenine nucleotides – Assists in export of mRNA from nucleus – Important in mRNA stability 3' mRNA 5' P P G P A Translation from nucleic acid language to amino acid language Radjewski The code • Code for ALL life! – strongest support for a common origin for all life • Code is redundant – several codons for each amino acid – 3rd base “wobble” Start codon AUG methionine Stop codons UGA, UAA, UAG Transfer RNA structure • “Clover leaf” structure – anticodon on “clover leaf” end – amino acid attached on 3 end Loading tRNA • Aminoacyl tRNA synthetase – enzyme which bonds amino acid to tRNA – bond requires energy • ATP AMP • bond is unstable • so it can release amino acid at ribosome easily Trp C=O OH OH Trp C=O O Trp H2O O activating enzyme tRNATrp anticodon tryptophan attached to tRNATrp AC C UGG mRNA tRNATrp binds to UGG condon of mRNA Building a polypeptide • Initiation – brings together mRNA, ribosome subunits, initiator tRNA • Elongation – adding amino acids based on codon sequence • Termination 3 2 1 – end codon Leu Val Met Met Met Met Leu Ala Leu Leu release factor Ser Trp tRNA 5' mRNA UAC AA U A UGC UG 3' E P A 5' UA C G A C A UG C U GA AU 5' 3' U A C GA C A U G C U G AA U 3' 5' U A C G A CG A A U AUG C U 3' ACC U GG UA A 3' Protein targeting • Signal peptide – address label Destinations: secretion nucleus mitochondria chloroplasts cell membrane cytoplasm etc… RNA polymerase DNA Can you tell the story? amino acids exon intron tRNA pre-mRNA 5' GTP cap mature mRNA poly-A tail large ribosomal subunit aminoacyl tRNA synthetase 3' polypeptide 5' small ribosomal subunit tRNA E P A ribosome Bacterial chromosome Protein Synthesis in Prokaryotes Transcription mRNA Psssst… no nucleus! Cell membrane Cell wall AP Biology Prokaryote vs. Eukaryote genes • Prokaryotes • Eukaryotes – DNA in cytoplasm – circular chromosome – naked DNA – no introns – DNA in nucleus – linear chromosomes – DNA wound on histone proteins – introns vs. exons introns come out! intron = noncoding (inbetween) sequence eukaryotic DNA exon = coding (expressed) sequence Translation in Prokaryotes • Transcription & translation are simultaneous in bacteria – DNA is in cytoplasm – no mRNA editing – ribosomes read mRNA as it is being transcribed Translation: prokaryotes vs. eukaryotes • Differences between prokaryotes & eukaryotes – time & physical separation between processes • takes eukaryote ~1 hour from DNA to protein – no RNA processing Any Questions?? What color would a smurf turn if he held his breath? AP Biology