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From Gene to Protein Question? How does DNA control a cell? By controlling Protein Synthesis. Proteins are the link between genotype and phenotype. Central Dogma DNA Transcription RNA Translation Polypeptide Explanation DNA - the Genetic code or genotype. RNA - the message or instructions. Polypeptide - the product for the phenotype. Genetic Code Sequence of DNA bases that describe which Amino Acid to place in what order in a polypeptide. The genetic code gives the primary protein structure. Genetic Code Is based on triplets of bases. Has redundancy; some AA's have more than 1 code. 20 amino acids: 64 codons Code Redundancy Third base in a codon shows "wobble”. First two bases are the most important in reading the code and giving the correct AA. The third base often doesn’t matter. Code Evolution The genetic code is nearly universal. Ex: CCG = proline (all life) Reason - The code must have evolved very early. Life on earth must share a common ancestor. Reading Frame and Frame Shift The “reading” of the code is every three bases (Reading Frame) Ex: the red cat ate the rat Frame shift – improper groupings of the bases Ex: thr edc ata tat her at The “words” only make sense if “read” in this grouping of three. Transcription Process of making RNA from a DNA template. Only one strand is used as a template. Where in the cell does transcription take place? Eukaryotes: nucleus Prokaryotes: cytoplasm Transcription Steps 1. 2. 3. 4. RNA Polymerase Binding Initiation Elongation Termination RNA Polymerase Enzyme for building RNA from RNA nucleotides. Binding Requires that the enzyme find the “proper” place on the DNA to attach and start transcription. Binding Is a complicated process Uses Promoter Regions (start region) on the DNA (upstream from the information for the protein) Initiation Actual unwinding of DNA to start RNA synthesis. Elongation RNA Polymerase untwists DNA 1 turn at a time. Exposes 10 DNA bases for pairing with RNA nucleotides. Elongation Enzyme moves 5’ 3’. Rate is about 60 nucleotides per second. Comment Each gene can be read by sequential RNA Polymerases giving several copies of RNA. Result - several copies of the protein can be made. Termination DNA sequence that tells RNA Polymerase to stop. Ex: AATAAA RNA Polymerase detaches from DNA after closing the helix. Final Product Pre-mRNA This is a “raw” RNA that will need processing. Modifications of RNA 1. 5’ Cap 2. Poly-A Tail 3. Splicing 5' Cap Modified Guanine nucleotide added to the 5' end. Protects mRNA from digestive enzymes. Recognition sign for ribosome attachment. Poly-A Tail 150-200 Adenine nucleotides added to the 3' tail Protects mRNA from digestive enzymes. Aids in mRNA transport from nucleus. RNA Splicing Removal of non-protein coding regions of RNA. Coding regions are then spliced back together. Introns Intervening sequences. Removed from RNA. Exons Expressed sequences of RNA. Translated into AAs. Result Introns - Function Left-over DNA (?) Way to lengthen genetic message. Old virus inserts (?) Way to create new proteins. Translation Process by which a cell interprets a genetic message and builds a polypeptide. Where in the cell does translation take place? Eukaryotes and prokaryotes: cytoplasm because that’s where the ribosomes are located. Materials Required tRNA Ribosomes mRNA Transfer RNA = tRNA Made by transcription. About 80 nucleotides long. Carries AA for polypeptide synthesis. Structure of tRNA Has double stranded regions and 3 loops. AA attachment site at the 3' end. 1 loop serves as the Anticodon. Anticodon Region of tRNA that base pairs to mRNA codon. Usually is a compliment to the mRNA bases, so reads the same as the DNA codon. Example DNA - GAC mRNA - CUG tRNA anticodon - GAC Ribosomes Two subunits made in the nucleolus. Made of rRNA (60%)and protein (40%). rRNA is the most abundant type of RNA in a cell. Large subunit Proteins rRNA Both sununits Large Subunit Has 3 sites for tRNA. P site: Peptidyl-tRNA site carries the growing polypeptide chain. A site: Aminoacyl-tRNA site holds the tRNA carrying the next AA to be added. E site: Exit site Translation Steps 1. Initiation 2. Elongation 3. Termination Initiation Brings together: mRNA A tRNA carrying the 1st AA 2 subunits of the ribosome Elongation Steps: 1. Codon Recognition 2. Peptide Bond Formation 3. Translocation Codon Recognition tRNA anticodon matched to mRNA codon in the A site. Peptide Bond Formation A peptide bond is formed between the new AA and the polypeptide chain in the P-site. After bond formation The polypeptide is now transferred from the tRNA in the P-site to the tRNA in the A-site. Translocation tRNA in P-site is released. Ribosome advances 1 codon tRNA in A-site is now in the P-site. Process repeats with the next codon. Termination Triggered by stop codons. Release factor binds in the A-site instead of a tRNA. H2O is added instead of AA, freeing the polypeptide. Ribosome separates. Prokaryotes Comment Polypeptide usually needs to be modified before it becomes functional. Examples Sugars, lipids, phosphate groups added. Some AAs removed. Protein may be cleaved. Join polypeptides together (Quaternary Structure).