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PROTEIN SYNTHESIS CENTRAL DOGMA OF MOLECULAR BIOLOGY: • DNA is used as the blueprint to direct the production of certain proteins What is a gene? • Gene = a segment of DNA coding for a RNA segment. – These RNA segments will be used to produce a polypeptide (structural or enzymatic protein) • Each strand of DNA can contain thousands of genes • Each gene has a precise beginning and an end Gene Expression • The DNA nucleotide sequence codes for the order in which amino acids are put together to form proteins • Every three nucleotides on the mRNA (codon) codes for a specific amino acid Genetic Code • 20 different amino acids but 64 possible codons – 43 = 64 – Some redundancy more then one codon codes for the same amino acid • Code is universal in almost all organisms Transcription • Information is transferred from DNA to RNA • Occurs in the nucleus Types of RNA • All three types of RNA are transcribed from DNA – Messenger RNA – carries the coded message from the DNA to the ribosome in the cytoplasm – Ribosomal RNA – reads the mRNA – Transfer RNA – transfers the correct amino acid to the ribosome Overview of Transcription • The segment of DNA that contains the gene for a specific protein or RNA that the cell wants to produce will unwind and the complementary RNA strand will be made by incorporation the RNA nucleotides Stages in Transcription • Each gene has a precise beginning known as the promotor region and an end known as the termination sequence Stages in transcription of RNA • Initiation – Transcription factors bind to the promoter region (TATA box) of the DNA – RNA polymerase then initiates transcription by binding to the transcription factor • Unwinds the DNA • Elongates the RNA segment Stages in transcription of RNA • Elongation – Nucleotides are added in the 5’ to 3’ direction by RNA polymerase – They form temporary hydrogen bonds with the DNA template – As the DNA helix reforms the RNA peels away Stages in transcription of RNA • Termination – At the end of the gene the termination sequence causes transcription to end – Pre-RNA segment dissociates from the DNA Terminator Post-transcriptional Modifications mRNA • 5’ cap – a guanine triphosphate is added – signal for ribosomal attachment in the cytoplasm • 3’ poly A tail – polyA polymerase adds ~250 “A” nucleotides to the end – protects RNA from being degraded by nucleases Post-transcriptional Modifications mRNA • Splicing – Exons – coding region – Introns – noncoding region • Cleaved out by snRNPs, and exons are spliced together Post-transcriptional Modifications Pre - mRNA Mature mRNA Post-transcriptional modifications rRNA • rRNA associates with proteins to form two subunits (40s and 60s) • Leaves the nucleus and enters the cytoplasm Post-transcriptional Modifications tRNA – Folds into a three dimensional structure (clover shaped) Translation • Going from the mRNA nucleotide code to amino acid code • mRNA is read by a ribosome (rRNA) to determine the sequence of amino acids • Occurs in the cytoplasm Translation • Players in Translation – mRNA strand – Ribosomes (rRNA) – tRNAs carrying amino acids – enzymes rRNA • rRNA has a mRNA binding site and three tRNA binding sites – A site (amino-acyl binding site) – P site (peptidyl binding site) – E site tRNA • Has an anticodon three base sequence that is complementary to a codon on the mRNA • 3’ end of the tRNA contains a binding site for a specific amino acid Stages of Translation • Initiation – mRNA binds to the 40s ribosome subunit – The initiator tRNA binds to the mRNA start codon (AUG) at the P site on the ribosome – The arrival of the 60s subunit completes the initiator complex Stages of Translation • Elongation – The next tRNA enters at the A site – The enzyme peptidyl transferase forms a peptide bond between the amino acid on the P site and the new amino acid on the A site. Stages of Translation: • Elongation – The ribosome then moves down the mRNA (translocation) – The tRNA that was at the A site is now at the P site and the empty tRNA that was at the P site now exits at the E site Charging of tRNA • Amino acids are floating freely in the cytoplasm • The enzyme amino-acyl tRNA synthetase attaches the amino acids to the 3’end of the tRNA • Requires ATP Stages of Translation • Termination – Elongation continues until a stop codon on the mRNA is reached (UAA, UAG, UGA) – The polypeptide is then released from the ribosome by a release factor Polysomes • Several ribosomes can simultaneously translate the same mRNA strand to make multiple copies of the same polypeptide Post-translational modifications to the Polypeptide • The start methionine is removed by the enzyme aminopeptidase • Protein will under go folding or modifications – Cleavage into smaller fragments or joined with other polypeptides – Chemical modifications: addition of carbohydrates or lipids – Transport to its destination Bigger Picture: What happens to these proteins once they are made? Summary Mutations and their consequences • Mutation = a change in the sequence of bases within a gene – Caused by a mistake during DNA replication (rare) – Or due to environmental factors called mutagens • Mutations can be somatic or germinal What is the main difference in the outcome of germinal mutation compared to the outcome of somatic mutation? Types of Mutations • Point mutations (substitutions) – change in a single nucleotide – Due to redundancy of the genetic code it may change the amino acid, it may not “wobble” – Silent mutations do not change the protein Normal hemoglobin DNA Sickle-cell hemoglobin mRNA Protein Glu Val Types of Mutations • Frame-shift mutation – caused by insertion or deletion of a nucleotide – Changes the reading frame of the codons, usually results in a non-functional protein Phe Leu Gly Ala Ala His Are all mutations bad? • Although mutations are sometimes harmful….. – They are also the source of the rich diversity of genes in the world – They contribute to the process of evolution by natural selection