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Molecular Biology DNA Expression CHAPTER 9 PART B DNA Expression: The Central Dogma oGenes contain specific sequences of bases coding the instructions for proteins ◦ In general one gene codes for one protein oThe basic process starts in the nucleus where enzymes transcribe the gene to make a strand of RNA. The RNA exits the nucleus through the nuclear pores. In the cytoplasm the RNA is translated into a sequence of amino acids (the building blocks of proteins) DNA RNA Transcription (Nucleus) Protein Translation (Cytoplasm) Transcription mRNA processing Assembly of RNA on unwound regions of DNA molecule mRNA rRNA tRNA proteins mature mRNA transcripts Translation At an intact ribosome, synthesis of a polypeptide chain at the binding sites for mRNA and tRNAs ribosomal subunits Convergence of RNAs mature tRNA cytoplasmic pools of amino acids, ribosomal subunits, and tRNAs Final protein Transcription: from DNA to mRNA oTranscription is the first step in gene expression oTranscription occurs in the nucleus in eukaryotes oThe base sequence of a gene in DNA is copied to make a single strand of RNA Transcription: from DNA to mRNA oDNA is unwound oOnly the part of the chromosome containing the gene is unwound oMakes a transcription bubble Transcription: from DNA to mRNA oRNA polymerase attaches to a binding site called a promoter oThe promoter is just in front of the gene (“up stream”) Transcription: from DNA to mRNA oRNA polymerase adds nucleotides one at a time using the DNA sequence as a template ◦ A-U, T-A, G-C, C-G Transcription: from DNA to mRNA oTranscription ends when the polymerase passes the end of the gene Eukaryotic mRNA Processing oOnce RNA is transcribed it will be modified before leaving the nucleus for the cytoplasm ◦ Introns are spliced out ◦ Eukaryotic genes contain ◦ Exons which are coding regions ◦ Introns which are non-coding regions ◦ Before the RNA leaves the nucleus the introns are snipped out leaving just the exons Eukaryotic mRNA Processing oOnce RNA is transcribed it will be modified before leaving the nucleus for the cytoplasm ◦ Protection from cytoplasmic RNA enzymes ◦ Poly A tail ◦ 50-300 adenines are added to the “tail” end (3’ end) ◦ 5’ “cap” ◦ A modified guanine is added to the “front” end (5’ end) Translation oTranslation is the second step in gene expression oTranslation occurs in the cytoplasm oThe base sequence in the mRNA is decoded into a sequence of amino acids in a protein Translation oThe Genetic Code ◦ The sequence of nucleotides/bases in the mRNA “spell” out the instructions for what protein the cell should make ◦ Different proteins determine the traits of cells and organisms ◦ Each set of three bases is one “word” or codon ◦ A codon indicates which amino acid to add to the protein ◦ Amino acids are protein monomers/subunits Translation oThe Genetic Code ◦ There are 64 different codons ◦ 61 codons code for 20 amino acids ◦ Thus most amino acids are encoded by more than one codon ◦ 1 codon is used as a start signal ◦ AUG (codes for methionine) ◦ 3 codons are used as a stop signal ◦ UAA, UGA, UAG Translation oThe Genetic Code 5’ ATG TCG GAC CTA 3’ 3’ TAC AGC CTG GAT 5’ 5’ AUG UCG GAC CUA 3’ (mRNA) met ser asp leu (start) Translation otRNA ◦ Each tRNA has two attachment sites ◦ One is an anticodon ◦ A triplet of bases complimentary to mRNA codons ◦ The other attaches to the amino acid specified by the codon Translation oInitiation ◦ Translation is initiated when the initiator tRNA binds the first AUG (start codon) of the mRNA and a small ribosomal subunit, then a large ribosomal subunit joins them ◦ Initiator tRNA’s anticodon base pairs with AUG ◦ Amino acid is methionine (met) ◦ Initiator tRNA + mRNA + small and large ribosomal subunits = initiation complex Translation oElongation 1. Initiator tRNA+met lines up in the P site of the ribosome ◦ ◦ Anti-codon matches AUG Carries methionine 2. Second tRNA+aa2 is placed in the A site of the ribosome ◦ The second tRNA’s anticodon matches the next three bases (codon) on the mRNA Carries the appropriate amino acid ◦ ◦ called aa2 to indicate it is the second amino acid 3. Peptide bond forms between met and aa2 4. Initiator tRNA is released from the P site and from its amino acid, met Translation oElongation 5. The second tRNA+aa2-met complex moves to the P site of the ribosome 6. Third tRNA+aa3 moves into the now vacant A site of the ribosome 7. Peptide bond forms between aa2 and aa3 ◦ Forms a polypeptide consisting of three amino acids ◦ Met-aa2-aa3 8. This process continues adding an amino acid for each codon 9. The polypeptide elongates Translation oTermination ◦ The ribosome encounters a stop codon in the mRNA ◦ There are no matching tRNAs for stop codons ◦ A release factor binds ◦ All the parts of the complex dissociate ◦ mRNA (can be used again) ◦ Small and large ribosomal subunits (can be used again) ◦ New polypeptide chain ◦ The new polypeptide will either stay in the cytoplasm or enter the rough endoplasmic reticulum of the endomembrane system Mutations oMutations are changes in the nucleotide sequence of DNA oThese can affect the sequence of amino acids in the encoded polypeptide thus affecting the function of the protein Mutations oCommon gene mutations ◦ Base-pair substitution ◦ A single base pair changes ◦ Affects one codon and thus one amino acid Mutations oCommon gene mutations ◦ Insertions ◦ One or more base pairs are added to the original DNA sequence ◦ Results in a reading frame shift Mutations oCommon gene mutations ◦ Deletions ◦ One or more base pairs are removed from the original DNA sequence ◦ Results in a reading frame shift Mutations oCommon gene mutations ◦ Frameshift (due to insertion or deletion) ◦ Shifts the 3-bases-at-a-time reading frame ◦ Changes the amino acid sequence from the point of mutation on. ◦ The cat ate the rat ◦ Tec ata tet her at (deletion) ◦ Thr eca tat eth era t (insertion) Mutations oMutation example: Sickle-cell anemia ◦ In the DNA sequence coding for the beta chain in hemoglobin there is a single base-pair substitution ◦ Hemoglobin carries oxygen through the blood ◦ When that DNA is transcribed to mRNA the mistake is copied ◦ When the mRNA is translated into a polypeptide one different amino acid is used ◦ Normal sequence: ◦ Mutated sequence: val-his-leu-thr-pro-glu-gluval-his-leu-thr-pro-val-glu- ◦ This one change disrupts the normal 3D folding of the protein ◦ Mis-shaped hemoglobin causes red blood cells to distort into a sickle shape under low oxygen conditions ◦ Causes clotting and disrupts blood circulation Summary oDNA expression ◦ Genes “code” for proteins and thus traits oTranscription oTranslation ◦ Genetic code oGene Mutations