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Deciphering the genetic code o Dr. Syndey Brenner estabilished mRNA as intermediate Discovered triplet nature of code Developed C. elegans as molecular model o Marshall Nirenburg Polyurdylic aci translation Codons associated with specific AA sequence The genetic code o Codon: 3 nudleotide base sequence o Use the bases A, G, C, and U with 64 possible combinations o Written 5’ to 3’ o Characteristics Specificity –unambiguous because a particular codon always codes for the same amino acid Universality – The specificity has been conserved from the early stages of evolution (only small differences) in how the code is translated Degeneracy –(or redundantcy) Each codon corresponds to a single amino acid, but a given amino acid may have more than one triplet coding for it. As a example, arginine is specified by six different codons. Methionine and tryptophan have just one codon. The wobble hypothesis: o The first two bases of a codon always form strong Watson Crick base-pair with the corresponding bases of anticodon and confer most of the coding specificity. o The first base of anticodon [5 ’to 3’ direction] or the 3rd base [3’ to 5’ direction] called the wobble base allows the single t-RNA to adopt more than one codon. The 3rd base [3’ to 5’ direction] of the codon leading to loose base pairing which is termed “wobble”. The wobble permits tRNA to read more than one codon with the maximum limits of three codons. o When the first base in the 5’-end in the anticodon is “U” then the third base in codon can be either “A” or “G”. The “U” forms strong Watson-Crick base pair with “A” and wobble pairing with “G”. Similarly if “G” present at 5’ ends of anticodon, then “G” forms Watson-Crick base pair with “C” and wobble base pair with “U”. o When “I” or some other modified base present at 5’-end of anticodon then t-RNA can recognize three different codons, all of which form a wobble base pairing at 3’ position of codon. The bases that can pair in this case are “A”, “U” and “C”. Non-overlapping and comma less – The code is read from a fixed starting point as a continuous sequence of bases taken three at a time ACGUGGAUACAU is read as AUG-UGG-AUA-CAU with no breaks or punctuation between the codons. o Important codons Start: AUG (sets the reading frame) Stop: UAG, UGA, UAA o Mutations Point mutation- a single base change Silent mutation – The codon containing the changed base codes for the same amino acid. Example, if the serine codon UCA is given a different third base – U – to become UCU it still codes for serine Missense mutations – The codon containing the changed base may code for a different amino acid. For example, if the serine codon UCA is given a different first base – C – to become CCA, it will now code for proline or an incorrect amino acid (missense) o Sickle cell anemia A point mutation in 6th codon of gene changes a GAG GTG During translation substitutes valine for normal glutamic acid Causes cells to be sickle shaped Hereditary blood disorder with co-dominant Nonsense mutation – The codon containing the changed base may become a termination codon. For example, if the serine codon UCA is given a different second base – A – to become UAA, the new codon causes termination of translation (a nonsense mutation) Frame-shift mutations – can change the reading frame if not 3 bases Insertion- an addition of one or more bases Deletion- a loss of one or more bases Trinucleotide repeat expansion- a 3 base repletion in tandom Huntington’s o CAG trinucleotide repeats which leads to defective protein that cause neurodegeneration Fragile X syndrome o Trinucleotde expanision in untranlated region causes decrease in overall amount of protein so neurons degernate during early development o Also seen I nmyotonic dystrophy Translation o mRNA, ribosome, charged tRNAs, accessory factors, GTP for energy o charging of the tRNA aminoacyl-tRNA synthetase required for attachement of AA to corresponding tRNAs two step reaction leading to the covalent attachment of the carboxy group of the AA to the 3’ end of the tRNA reaction requires ATP proof-reading and editing activity o the ribosome small ribosomal unit: bind mRNA and is responsible for the accuracy of translation by facilitating correct base pairing between codon and mRNA large subunit: catalyzes formation of peptide bonds that link the AA in growing polypeptide chain 3 sites of interaction between ribosome, tRNA, and mRNA A site: aminoacyl site where incoming charged tRNA attaches P site: the peptidyl site where the peptide bond is formed E site: exit site depleted tRNA is released o Prokaryotes Can have multiple ribosome biding sites: Shine-Dalgarno sequences Recognition is facilitated by IF-2-GTP in prokaryotes and eIF-2-GTP (plus additional eIFs) in eukaryotes In bacteria and in mitochondria the initiator tRNA carries an N-formylated methionine GTP is hydrolyzed and 50S subunit is recruited with loss of IF’s. Note – in prokaryotes three initiation factors are known (IF-1, IF-2, IF-3) whereas eukaryotes have over 10 initiation factors (eIF) The large ribosomal subunit then joins the complex, and a functional ribosome is formed with the charged initiating tRNA in the P site, and the A site is empty Formation of the peptide bond is catalyzed by peptidyltransferase, an activity intrinsic to the 23S rRNA found in the 50S subunit The ribosome then advances three nucleotides toward the 3’-end of the mRNA. This process is known as translocation and in prokaryotes requires participation of EF-G-GTP and GTP hydrolysis (eukaryotes cells use EF-2-GTP) Attenuation Excess tryptohphan: transcription terminated Tryptophan starved: transcription not terminated Transcription and translation can be coupled in bacterial cells Leader sequence o POL o Eukaryotes process is more complex compared to prokaryotes. also a site for regulation of protein synthesis Initiation of translation involves formation of a complex composed of : methionyl-tRNAi met mRNA a ribosome Initiation: methionyl-tRNAi met initially forms a complex with the protein eukaryotic initiation factor 2 (eIF2) which binds GTP (This complex binds to the small (40S) ribosomal subunit) The cap at the 5’-end of the mRNA binds an initiation factor known as the cap-binding protein (CBP or eIF4F)---CBP contains a number of small subunits, including eIF4E Several other eIFs join, and the mRNA then binds to the eIFs-MettRNAiMet-40S ribosomal complex In this reaction, hydrolysis of ATP is required because a helicase is needed to unwind the hairpin loop in the mRNA and scans the mRNA until it locates the AUG start codon GTP is hydrolyzed, the initiation factors are released, and the large ribosomal (60S) subunit binds (The ribosome is now complete). o The 80S eukaryotic ribosome contains one small (40S) and one large (60S) subunit and it has two binding sites for tRNA, known as the P and A sites. Elongation When Met-tRNA is bound to the P site, the mRNA codon in the A site determines which aminoacyl –tRNA will bind to that site In eukaryotes, the incoming aminoacyl-tRNA first combines with elongation factor EF1a containing bound GTP before binding to the mRNA ribosome complex (EF1 is the GTP-binding a-subunit of a heterotrimeric G-protein which is activated for association with other proteins when it contains GTP) When the aminoacyl-tRNA –EF1-GTP complex binds to the A site, GTP is hydrolyzed to GDP. This releases EF1-GDP from the aminoacyl –tRNA ribosomal complex; proteins synthesis continues Free EF1-GDP re-associates with the EF1-subunits, and GDP is released GTP binds and the bg-subunit dissociates. Then, EF1-GTP is ready to bind another aminoacyl –tRNA Contrast: Elongation in prokaryotes is similar – except that the corresponding factor for EF1 is named EF-Tu and the associating elongation factors are called EF-Ts instead of EF1. Termination at the stop codon Hormone regulation of protein synthesis Insulin (hormone) can regulate protein translation: o Stimulates via activation of eIF4E (initiation factor) o Normally, eIF4E is bound to an inhibitor protein, 4EBinding factor o When insulin binds to it’s receptor that results in phosphorylation of 4EBP and that releases eIF4E for initiation of protein synthesis Polyribosome (polysomes) Where multiple copies of polypeptides can be made at once Post-translational modifications Various enzymatic modifications can occur to affect protein function or structure Proteolytic cleavage removes the terminal 5’ Met, and other proteases may cleave the protein. Nine amino acid modifications o Acetylation o Methylation o Hydroxylation o Carboxylation o Fatty acylation o Prenylation o ADP ribosylation o Phosphorylation/dephosphorylation o Glycosylation N-glycosylation occurs in the endoplasmic reticulum and O-glycosylation in the Golgi Proteins destined for secretion o Initially functionally inactive o Cleavage can occur or placed in secretory vesicles o Zymogens are inactive precursors of secreted proteins and become activated through cleavage after reaching a site of action (example: pancreatic zymogen, trypsinogen, activated to trypsin in the small intestine. Chaperones- mediate posttranslational folding Foldases- support folding of proteins in ATP-dependent manner Holdases- bind folding intermediates to prevent aggregation Diptheria toxin mechanism of action Diphtheria toxin is a bacterial AB toxin whose gene is encoded by a virus that infects Corynebacterium diphtheriae. Diphtheria toxin B-subunit binds to a cell surface receptor, facilitating the entry of the A-subunit into the cell. The A-subunit catalyzes a reaction where the ADP-ribose (ADPR) portion of NAD is transferred to EF2 (ADP-ribosylation). ADPR is attached covalently to a post-translationally modified histidine residue, known as diphthamide. ADP-ribosylation of EF2 inhibits protein synthesis, leading to cell death Antibiotic mechanism of action