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From Gene to Protein CONCEPT 1: Genes Specify Proteins via Transcription and Translation Gene Expression – process by which DNA directs the synthesis of proteins (or RNAs)-old idea: one gene-one enzyme hypothesis – proposed by Beadle and Tatum from mutant mold experiments. Function of a gene is to dictate production of specific enzyme-newer idea: one gene-one polypeptide hypothesis-most accurate: one gene-one RNA molecule (which can be translated into a polypeptide) Flow of Genetic Information: DNA à RNA à Protein-central dogma: DNA à RNA à Protein-transcription: DNA à RNA-translation: RNA à Protein (ribosome = site of translation) Flow of genetic information in prokaryotes vs. eukaryotesProkaryotes: DNA à RNA à Protein all happens in the cytoplasm DNA à mRNA à Protein synthesized on ribosomeEukaryotes: DNA à RNA à Protein(DNA à pre-mRNA à mRNA) transcription and RNA processing happens in the nucleus, the mRNA is exported and à Protein synthesized by ribosomes in cytoplasm during translation comparison of DNA and RNA-DNA: nucleic acid composed of nucleotides. Double-stranded. Deoxyribose = sugar. Thymine. Template for individual.-RNA: nucleic acid composed of nucleotides. Single-stranded. Ribose = sugar. Uracil. Many different roles. RNA plays many roles in the cell1. pre-mRNA = precursor to mRNA, newly transcribed and not edited2. mRNA = edited version; carries the code from DNA that specifies amino acids3. tRNA = carries a specific amino acid to ribosome based on its anticodon to mRNA codon4. rRNA = makes up 60% of the ribosome; site of protein synthesis5. snRNA = small nuclear RNA; part of a spliceosome; has structural and catalytic roles6. srpRNA = a signal recognition particle that binds to signal peptides7. RNAi = interference RNA; a regulatory molecule8. Ribozyme = RNA molecule that functions as an enzyme The Genetic Code-for each gene, one DNA strand is the template strand-mRNA (5’à3’) complementary to template-mRNA triplets (codons) code for amino acids in polypeptide chain-64 different codon combinations-redundancy: 1+ codons for each of 20AAs-reading frame: groups of 3 must be read in correct groupings-this code is universal: all life forms use the same code CONCEPT 2: Transcription is the DNA-directed synthesis of RNA Transcription unit – stretch of DNA that codes for a polypeptide or RNA (eg. tRNA, rRNA) RNA polymerase:-separates DNA strands and transcribes mRNA-mRNA elongates in 5’à3’ direction-uracil (U) replaces thymine (T) when pairing to adenine (A)-attaches to promoter (start of gene) and stops at terminator (end of gene) 1. Initiation-Bacteria: RNA polymerase binds directly to promoter in DNA-Eukaryotes: TATA box = DNA sequence (TATAAAA) upstream from promoter. Transcription factors must recognize TATA box before RNA polymerase can bind to DNA promoter 2. Elongation-RNA polymerase adds RNA nucleotides to the 3’ end of the growing chain (A-U, G-C)-as RNA polymerase moves, it untwists DNA, then rewinds it after mRNA is made 3. Termination-RNA polymerase transcribes a terminator sequence in DNA, then mRNA and polymerase detach-it is now called pre-mRNA for eukaryotes-in prokaryotes the mRNA is ready for use CONCEPT 3: Eukaryotic cells modify RNA after transcription Additions to pre-mRNA:-5’ cap (modified guanine) and 3’ poly-A tail (50-520 As) are added-help export from nucleus, protect from enzyme degredation, attach to ribosomes RNA splicing:-pre-mRNA has introns (noncoding sequences) and exons (codes for amino acids)-splicing = introns cut out, exons joined together-small nuclear ribonucleoproteins = snRNPs -snRNP = snRNA + protein -pronounces ‘snurps’ -recognize splice sites-snRNPs join with other proteins to form a spliceosome-spliceosomes catalyze the process of removing introns and joining exons-Why have introns? -some regulate gene activity-alternative RNA splicing: produce different combinations of exons – so one gene can make more than one polypeptide. -20,000 genes à 100,000 polypeptides CONCEPT 4: Translation is the RNA-directed synthesis of a polypeptide Components of translation:1. mRNA = the message2. tRNA = the interpreter3. ribosome = the site of translation tRNA:-transcribed in the nucleus-specific to each amino acid-transfer AA to ribosomes-anticodon: pairs with complementary mRNA codon-base-pairing rules between 3rd base of codon and anticodon are not strict. This is called wobble.-Aminoacyl-tRNA-synthetase: enzyme that binds tRNA to specific amino acid Ribosomes:-ribosome = rRNA + proteins-made in nucleolus-has 2 subunits (large subunit and small subunit)-active sites: A site – holds AA to be added P site – holds growing polypeptide chain E site – exit site for tRNA Process of Translation1. Initiation-small subunit binds to start codon (AUG) on mRNA-tRNA carrying Met attaches to P site-large subunit attaches 2. Elongation-codon recognition: tRNA anticodon matches codon in A site-peptide bond formation: AA in A site forms bond with peptide in P site-translocation: tRNA in A site moves to P site; tRNA in P site moves to E site (then exits)-repeats over and over 3. Termination-Stop codon reached and translation stops-release factor binds to stop codon;polypeptide is released-ribosomal subunits dissociate Polyribosomes = a single mRNA can be translated by several ribosomes at the same time Protein folding-during synthesis, polypeptide chain coils and folds spontaneously-Chaperonin: protein that helps polypeptide fold correctly Types of Ribosomes:-Free ribosomes – synthesize proteins that stay in cytosol and function there-Bound ribosomes (to ER) – make proteins of endomembrane system (nuclear envelope, ER, Golgi, lysosomes, vacuoles, plasma membrane) and proteins for secretion – use signal peptides to target location Cellular “zip codes”-signal peptide: 20 AA at leading end of polypeptide determines destination-signal recognition particle (SRP): brings ribosome to ER CONCEPT 5: Point mutations can affect protein structure and function Mutations = changes in the genetic material of the cell-large scale mutations: chromosomal; always cause disorders or death -nondisjunction, translocation, inversions, duplications, large deletions-point mutations: alter 1 base pair of a gene 1. base-pair substitutions – replace one with another --missense: different amino acid --nonsense: stop codon, not amino acid --silent: no effect (results in same amino acid) 2. Frameshift – mRNA read incorrectly; nonfunctional proteins --caused by insertions or deletions --can cause extensive missense, premature terminationex: sickle cell disease is due to a point mutation, forms a mutant protein due to a single AA change. Sickle cell hemoglobin forms long inflexible chains. Disease results COMPARISON: Prokaryotes vs. EukaryotesProkaryotes:-transcription and translation both in cytoplasm-DNA/RNA in cytoplasm-RNApol binds directly to promoter-transcription makes mRNA (not processed)-no introns Eukaryotes:-transcription in nucleus, translation in cytoplasm-DNA in nucleus, RNA travels in/out nucleus-RNApol binds to TATA box and transcription factors-transcription makes pre-mRNA à RNA processing à final RNA
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