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Chapter 10B: Gene Expression 1. Overview of Gene Expression 2. Transcription 3. The Genetic Code 4. Translation 1. Overview of Gene Expression Overview of Gene Expression DNA (genetic info) transcription (in nucleus) RNA (copy) translation (via ribosomes in cytoplasm) Protein 1 Gene Expression The expression of a gene into an actual protein occurs by 2 basic processes: 1) Transcription of a gene into RNA • RNA is a nucleic acid very similar to DNA (RNA uses “U” instead of “T”) • this is essentially creating a “photocopy” of the gene • occurs in the nucleus 2) Translation of the RNA transcript into protein • accomplished by ribosomes, in the cytoplasm Comparison of DNA & RNA RNA DNA • sugar = Ribose • sugar = Deoxyribose • single-stranded • double-stranded • A, C, G & U (uracil) • A, C, G & T (thymine) 2. Transcription 2 Transcription Transcription of gene to make an RNA copy is much like DNA replication except for the following: RNA nucleotides RNA polymerase A T C C A A U T G A G G T Direction of transcription Newly made RNA G C A U C C A G U T • catalyzed by RNA polymerase T C T A • only 1 strand is used as a template C • produces complementary RNA C T A A • involves only 1 gene Template Strand of DNA • RNA is released, DNA “zips” back up! *results in a copy of one DNA strand* The Process of Transcription 3 Functions of RNA made by Transcription * 1) mRNA* • copy of gene 2) tRNA • delivers AAs to ribosomes 3) rRNA *provides genetic info for translation • part of ribosomes 3. The Genetic Code How are Genes related to DNA? Genes are segments of DNA that code for a particular protein (or RNA molecule) • the human genome contains ~3 billion base pairs (bps) and ~25,000 genes • almost all genes encode proteins • when we talk about “genes” we will focus on those that express proteins ( the “end products” for a small percentage of genes are special types of RNA molecules) 4 What does DNA actually code for? In other words, “How do genes encode proteins”? • recall that proteins are linear polymers made of the 20 different amino acids ***genes need simply to encode the identity of each amino acid in a given protein*** • i.e., genes must be capable of encoding 20 different amino acids and their order in a protein • although DNA contains only 4 “letters” (i.e., nucleotides), this is more than sufficient… The Genetic Code Each amino acid in a protein is specified by 3 nucleotide sequences called codons • each of the 20 amino acids is coded for by a unique set of codons: e.g. ATG = methionine (start codon) GGN = glycine CAA or CAG = glutamine • there are 64 possible “codon” triplets (4 x 4 x 4) • more than enough to encode 20 amino acids and the signal to “stop” or end the protein (TGA, TAA or TAG) The Genetic Code Second base If the DNA sequence is: CATGCCTGGGCAATAG Third base First base (transcription) The mRNA copy is: CAUGCCUGGGCAAUAG (translation) The polypeptide is: *Met-Pro-Gly-Gln-(stop) all proteins begin w/Met 5 From DNA to RNA to Protein DNA strand Transcription RNA Codon Translation Polypeptide Amino acid The Effects of Mutation MUTATION: any change in DNA sequence Normal gene A U G A A G U U U G G C G C A mRNA Met Protein Lys Phe Gly Ala Base substitution A U G A A G U U U A G C G C A Met Lys Phe Ser • deletions, insertions cause a change in reading frame (frameshift) Ala U Missing Base deletion A U G A A G U U G G C G C A U Lys Met Leu Ala His Sickle Cell Anemia is due to a Base Substitution Normal hemoglobin DNA C T T G A A mRNA Mutant hemoglobin DNA C A T G U A mRNA Normal hemoglobin Glu Sickle-cell hemoglobin Val 1 nucleotide changes 1 amino acid resulting in a misfolded hemoglobin protein (clump together in RBCs > sickle shape) 6 4. Translation Overview of Translation Translation occurs in the cell cytoplasm and involves: Messenger RNA (mRNA) • copy of the gene to be expressed into protein Ribosome • structure made of rRNA and protein that catalyzes the polymerization of amino acids into a polypeptide Transfer RNAs (tRNAs) • deliver amino acids to ribosomes during translation Messenger RNA (mRNA) Start of genetic message AUG… 5’ End 3’ UGA or UAA or UAG 7 Ribosome Structure tRNA-binding sites Large subunit mRNA binding site Small subunit tRNA Structure & Function Amino acid attachment site Hydrogen bond RNA polynucleotide chain mRNA codons will “base pair” with a complementary anticodon in a tRNA… Anticodon Ribosomes translate mRNA to Protein Next amino acid to be added to polypeptide in the cytoplasm Growing polypeptide tRNA mRNA 5’ 3’ Codons 8 Translation (“protein synthesis”) The building of a polypeptide, 1 amino acid at a time, by ribosomes using info in mRNA: • ribosomes bind directly to mRNA, “read” codon by codon • ribosomes always start at AUG (methionine) • translation also involves tRNAs, each of which is attached to 1 of the 20 amino acids (AAs) • ribosomes match the right tRNA (via the anticodon) with the right codon in the mRNA, then add its AA to the growing protein Initiation of Translation M et M et Large ribosomal subunit Initiator tRNA P site 1 Start codon mRNA Small ribosomal subunit A site 2 • small ribosomal subunit, mRNA, and intiator tRNA (tRNAMet) assemble first (at P site) • large ribosomal subunit then joins the complex after which a 2nd tRNA enters the A site… • tRNA with complementary anticodon Amino acid Polypeptide A site P site Anticodon mRNA Elongation of Translation Codons 1 Codon recognition 1) tRNA with complementary anticodon enters A site mRNA movement Stop codon 2 Peptide bond formation New peptide bond 3 Translocation 2) peptide bond forms between amino acids from each tRNA 3) ribosome shifts (translocates) to next codon, process repeats… 9 Termination of Translation New peptide bond forming Growing polypeptide Elongation A succession of tRNAs add their amino acids to the polypeptide chain as the mRNA is moved through the ribosome, one codon at a time. Codons mRNA Polypeptide Termination The ribosome recognizes a stop codon. The polypeptide is terminated and released. Stop codon General Steps of Translation 1) ribosome begins translation at AUG of mRNA 2) ribosome binds 2 tRNA-AAs, 2 codons at a time • i.e., tRNAs with anti-codons complementary to the mRNA codons 3) ribosome then catalyzes peptide bond formation between the amino acids attached to each tRNA 4) ribosome shifts 3 nucleotides (1 codon) on mRNA and repeats the process 5) this continues until the ribosome reaches a “stop” codon which causes translation to end Summary of Gene Expression Transcription DNA 1 mRNA RNA polymerase mRNA is transcribed from a DNA template. Translation Amino acid 2 Each amino acid attaches to its proper tRNA with the help of a specific enzyme and ATP. Enzyme ATP tRNA Anticodon Large ribosomal subunit Initiator tRNA 3 Initiation of Translation The mRNA, the first tRNA, and the ribosomal sub-units come together. Start Codon mRNA Small ribosomal subunit 10 Key Terms for Chapter 10B • mRNA, tRNA, rRNA • transcription, RNA polymerase • codon, anti-codon • genetic code • mutation: substitution, deletion, insertion • reading frame, frameshift • translation, ribosome Relevant Review Questions: 1, 3-5, 7, 9 11