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Ch 17 Gene Expression II: Translation mRNA->Protein LE 17-4 Gene 2 Review DNA molecule Gene 1 Gene 3 DNA DNA strand (template) 5 3 TRANSCRIPTION RNA mRNA 5 3 Codon TRANSLATION Protein Protein Amino acid LE 17-4 Gene 2 Review DNA molecule Gene 1 Gene 3 DNA DNA strand (template) 5 3 TRANSCRIPTION RNA mRNA 5 3 Codon TRANSLATION Protein Protein Amino acid Cracking the Code • 64 codons – decoded by the mid-1960s •Genetic code –redundant but not ambiguous; no codon specifies more than one amino acid (but one amino acid may have >1 codon) •Codons – must be read in the correct reading frame in order for the specified polypeptide to be produced LE 17-5 Second mRNA base Codon Table Third mRNA base (3 end) Genetic Code Find an example of redundancy in the genetic code. Which amino acid does not have redundant codons? Is there a pattern to redundant codons? Evolution of the Genetic Code • Genetic code – nearly universal: shared by the simplest bacteria, plants, fungi and animals • Genes can be transcribed and translated after being transferred from one species to another Mechanism of Translation • Ribosomes - Bind messenger (mRNA) - Attract transfer RNA (tRNA) to mRNA - tRNA covalently linked to specific amino acid (aa-tRNA) -Complementary basepairs form between mRNA and aatRNA (codon-anticodon interactions) -Enzyme in ribosome catalyzes peptide bond between amino acids - -> polypeptide chain grows LE 17-14a tRNA structure 3 Amino acid attachment site 5 ~ 80 nt long Three different schematics Hydrogen bonds Anticodon Two-dimensional structure Amino acid attachment site 5 In what ways do they convey the same and different information? 3 Hydrogen bonds 3 Anticodon Three-dimensional structure 5 Anticodon Symbol used in this book LE 17-13 Amino acids Polypeptide tRNA with amino acid attached Ribosome tRNA Anticodon Codons 5 mRNA 3 Accurate translation requires two steps 1. a correct match between tRNA and an amino acid - Catalyzed by aminoacyl-tRNA synthetase 2. a correct match between the tRNA anticodon and an mRNA codon LE 17-15 Amino acid Aminoacyl-tRNA synthetase (enzyme) 1. Pyrophosphate Phosphates tRNA AMP Aminoacyl tRNA (an “activated amino acid”) Ribosomes • Facilitate specific coupling of anticodons with codons Draw • Ribosomal structure – Two ribosomal subunits (large and small) • Made of proteins (ribosomal proteins) and ribosomal RNA (rRNA) Form binding sites for mRNA and aa-tRNA LE 17-16a tRNA molecules Growing polypeptide Exit tunnel Large subunit E P A Small subunit 5 3 mRNA Computer model of functioning ribosome LE 17-16b Schematic model showing binding sites on ribosome P site (Peptidyl-tRNA binding site) A site (AminoacyltRNA binding site) E site (Exit site) E mRNA binding site P A Large subunit Small subunit LE 17-16c Amino end Growing polypeptide Next amino acid to be added to polypeptide chain E tRNA mRNA 5 3 Codons Schematic model with mRNA and tRNA Ribosome translates 5’ to 3’ on mRNA. Polypeptide chain grows amino end first, carboxyl end last. Building a Polypeptide • The three stages of translation: – Initiation – Elongation – Termination •All three stages require protein translation factors Ribosome Association and Initiation of Translation 1. Small ribosomal subunit binds mRNA and special initiator tRNA (met-tRNAi) (carries the amino acid methionine) 2. Small subunit scans along the mRNA until first start codon (AUG). 3. Initiation factors bring in large subunit initiator tRNA occupies the P site. LE 17-5 Second mRNA base Codon Table Genetic Code Third mRNA base (3 end) Memorize Start Codon LE 17-17 Large ribosomal subunit P site Initiator tRNA GTP GDP E A mRNA 5 3 5 3 Start codon mRNA binding site Small ribosomal subunit Translation initiation complex Elongation of the Polypeptide Chain - Amino acids are added one by one to the preceding amino acid -Elongation factors facilitate - codon recognition - peptide bond formation - translocation LE 17-18 1. Recognition Amino end of polypeptide E 3 mRNA Ribosome ready for next aminoacyl tRNA P A site site 5 2 GTP 2 GDP E E P A P A GDP GTP 3. Translocation 2. Peptide bond formation E P A Termination of Translation - Occurs when stop codon in mRNA reaches A site of ribosome - A site accepts protein called release factor -Release factor causes addition of water molecule instead of amino acid - Polypeptide released, ribosomal subunits dissociate and fall off mRNA LE 17-5 Second mRNA base Codon Table Genetic Code Third mRNA base (3 end) Memorize Stop Codons LE 17-19 Release factor Free polypeptide 5 3 3 3 5 5 Stop codon (UAG, UAA, or UGA) When a ribosome reaches a stop codon on mRNA, the A site of the ribosome accepts a protein called a release factor instead of tRNA. The release factor hydrolyzes the bond between the tRNA in the P site and the last amino acid of the polypeptide chain. The polypeptide is thus freed from the ribosome. The two ribosomal subunits and the other components of the assembly dissociate. Let’s translate a mRNA… 5’ cgaggucaaugcccuauguuuagccc 3’ Bracket each codon in the open reading frame (ORF). Write the amino acid below each codon. What is the anticodon for the second codon in the ORF? I’m complicated but once you get to know me I’m really pretty nice. Any questions? 5’ 3’ Can a transcript (mRNA) be translated by multiple ribosomes simultaneously? Polyribosomes • -a single mRNA (transcript) is translated by many ribosomes simultaneously • mRNA+ bound ribosomes= polyribosomes or polysome • Allows fast synthesis of many copies a polypeptide LE 17-20 Polyribosome or Polysome Incoming Completed polypeptides Growing polypeptides ribosomal subunits Start of mRNA (5 end) End of mRNA (3 end) An mRNA molecule is generally translated simultaneously by several ribosomes in clusters called polyribosomes. Ribosomes mRNA 0.1 mm This micrograph shows a large polyribosome in a prokaryotic cell (TEM). Consider: When a eukaryotic message is transcribed, processed and transported to the cytosol, is it immediately translated into protein? When would a cell need a polypeptide immediately? When would a cell want to delay translation? Examples? What strategy could one use to determine whether a mRNA was being actively translated? Hint: consider mass Subject cell homogenate to differential centrifugation -Heavy polysomes will pellet -Light untranslated mRNA in supernatant Polysomes in Prokaryotes Where and when are transcripts translated in prokaryotes? Coupled transcription and translation LE 17-22 RNA polymerase DNA mRNA Polyribosome RNA polymerase Direction of transcription 0.25 mm DNA Polyribosome Polypeptide (amino end) Ribosome mRNA (5 end) Targeting Polypeptides to Specific Locations In eukaryotes, what are the two populations of ribosomes? Free, soluble in cytosol synthesize soluble proteins Bound to rER - synthesize secreted or membrane bound proteins - tagged with signal peptide at amino end LE 17-21 Signal peptide targets polypeptides to ER final polypeptide destined for secretion or membrane Ribosomes mRNA Signal peptide Signalrecognition particle SRP (SRP) receptor ER membrane Signal peptide removed Protein protein CYTOSOL ER LUMEN Translocation complex Is the molecular weight of a secreted protein different than the predicted translation product of its mRNA? Effect of mutations on gene expression What is a mutation? Any change in the genetic material of a cell or virus Types of mutations Point: a single nucleotide change -substitution gcca->gcga -deletion gcca->gca -insertion gcca->gacca Also, breaks, translocations, inversions as reviewed previously LE 17-23 What kind of mutation? substitution Wild-type hemoglobin DNA 3 Mutant hemoglobin DNA 5 3 mRNA 5 mRNA Transcribe into mRNA 5 3 Normal hemoglobin 5 3 Sickle-cell hemoglobin Translate into protein LE 17-5 Second mRNA base Codon Table Genetic Code Third mRNA base (3 end) Memorize Start Codon Substitutions • Missense mutations – Change codon to encode a different amino acid • Nonsense mutations – Change codon to encode a stop codon nearly always leading to a nonfunctional protein Missense mutations are more common. Why? LE 17-24 Wild type mRNA 5 Protein 3 Stop Amino end Carboxyl end Base-pair substitution No effect on amino acid sequence U instead of C Substitutions Neutral Stop Missense A instead of G Change in amino acid Stop Nonsense U instead of A Premature termination Stop Insertions and Deletions • Alters reading frame ->frameshift mutation • Often more devastating than substitutions LE 17-25 Wild type mRNA 5 Protein 3 Stop Carboxyl end Amino end Base-pair insertion or deletion Addition frameshift Extra U Stop Deletion frameshift Missing Insertion or deletion of 3 nucleotides Missing Stop Source of Mutations • From spontaneous mutations: occur during DNA replication, recombination, or repair • From mutagens are physical or chemical agents that can cause mutations What is a gene? revisiting the question • A gene is a region of DNA whose final product is either a polypeptide or an RNA molecule LE 17-26 TRANSCRIPTION DNA 3 5 RNA polymerase RNA transcript RNA PROCESSING Exon RNA transcript (pre-mRNA) Intron Aminoacyl-tRNA synthetase NUCLEUS CYTOPLASM FORMATION OF INITIATION COMPLEX Amino acid AMINO ACID ACTIVATION tRNA mRNA Growing polypeptide Activated amino acid 3 A P E Ribosomal subunits 5 TRANSLATION E A Codon Ribosome Anticodon