MCB 150 Merit 10/18/12

Lecture # 19 Translation
1. Codons are part of the nucleotide sequence of a mRNA molecule a. They determine the order in which amino acids are linked together to form a protein b. BUT amino acids do NOT directly contact the Mrna i. they are held onto mRNA by tRNA molecules 2. tRNAs act as bridges between amino acids and the mRNA a. tRNAa are single stranded b. short (75-80 nucleotide) single stranded RNA, but it folds up on itself by Intrastrand base pairing i. 2-dimensional structure resembles a clover leaf ii. two most ULTRA important region are at opposite ends, in regions of unpaired nucleotides 1. Anti-codon site 2. Amino acid attachment site 3. tRNA structure: a. REMEMBER : it is a single stranded Rna b. It has an amino attachment site at the top or at the end of this tRNA structure. i. It will always have the sequence [CCA] read from 53 direction!! c. It has a structure that resembles a clover leaf in its 2-D structure d. Hydrogen bonds are holding together the base pairs e. There is an anticodon site, at the other end of the structure i. It is complementary to the codon in mRNA f. Its 3-D structure resembles a power drill i. If it has a little ball it means that it is a charged tRNA 1. Which means that it has an amino acid attached into its amino acid attachment site ii. If it doesnt have one, it is an uncharged tRNA 1. No amino acid attached 4. The binding of an amino acid to a tRNA molecule is VERY specific a. requires input of energy provided by hydrolysis of ATP to AMP and pyrophosphate (PPi) i. AMP when broken two phosphates b. reaction catalyzed by enzymes called Aminoacyl tRNA Synthetases (AARS) i. a different aminoacyl tRNA synthetase for each Amino Acid 1. e.g. histidyl tRNA synthetase adds His to its tRNA

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2. Therefore there are 20 different kinds of enzymes ii. Each enzyme recognizes both ends of tRNA, which enables it to identify if this is a tRNA that it is allowed to add its amino acid to it. 1. The process is called CHARGING 2. They can recognize the 3-D structure of anti-codons 3. Based on what they recognize in the anti-codon site they will either go or not go to link at the amino acid site a. Therefore they consult first with the anti-codon site Recognition of tRNA by an aminoacyl tRNA synthetase: a. The 3-D structure resembles a power drill b. The amino acid reads the anti-codon and then it goes and links to the amino acid end tRNA charging by Aminoacyl tRNA synthetase: a. It is always a 3 carbon adenine base b. There can be two types of classes of tRNA that an amino acid can covalently bond to either the 2 or 3 carbon hydroxide group. Site of protein synthesis = Ribosomes a. Platform of protein synthesis i. Universal mostly, structures very different b. RNA + Proteins= Ribosomes c. complexes of ribosomal RNA (rRNA) and ribosomal proteins d. found in cytoplasm of prokaryotes & eukaryotes e. functionally identical, but structurally different between bacteria and eukaryotes i. differences are important, because many antibiotics work by specifically inhibiting bacterial ribosomes 1. What if the antibiotic worked on both euk. & bact. ribosomes? a. Then it will attack all of your ribosomes depleting you of your resources, it is key for an antibiotic to only identify bacteria ribosomes. Ribosome Structure: a. A Prokaryotic 70S ribosomes will contain i. One or more pieces of rRNA and proteins (small polypeptides) classified as 1. 23S and 5S rRNAs (34 proteins)- Large subunit [50S] 2. 16 S rRNA (21 Proteins) Small subunit [30S] b. Eukaryotic 80 S ribosome i. ~45 proteins - Large subunit [60S] 1. Take less space ii. ~30 proteins small subunit [40S] What is required for Initiation of Translation?

a. mRNA Infor b. small ribosomal subunit c. first charged tRNA (always charged with Met*) i. Or some variant of Met, especially in bacteria [fMet] d. accessory proteins called Initiation Factors Help e. energy in form of GTP (equivalent in E to ATP) 10. AUG is called the Initiator Codon a. it begins or initiates all protein chains b. codes for special modified Met (N-formyl-methionine, or fMet) in bacteria, unmodified Met in eukaryotes c. AUG can be found in other codons, but regular Met is added in bacteria (fMet only starts chain) d. archae have Met, not fMet, as first amino acid e. In eukaryotes is always going to start with Met. f. Archae are biochemically more similar to eukaryotes 11. Translation Initiation:

a. Due to the presence of fMet we can conclude that this is bacteria translation b. Notice nomenclature of tRNA (superscript indicates what AA it should carry) i. Important to remember that binding of lg. subunit requires energy ii. Notice that large subunit has room for 2 hybridized tRNAs A and P sites 1. A = Aminoacyl site 2. P = Peptidyl site c. How does a ribosome distinguish initiator codon from an internal methionine codon?

i. There could be a lot of bases ii. The method if distinguishing differs in bacteria and eukaryotes iii. In bacteria 1. The sequence is called Shine-Delgarno or Ribosome Binding Site (RBS) a. This is already hyberdized in Mrna b. Aligns initiator codon with initiator tRNA c. It is a conserved and consensus region in bacteria i. 53 AGGAGGU d. Bacterial mRNAs can have multiple RBS (Ribosomes Binding Sites) i. Bacterial mRNAs that carry information for multiple protein products are called POLYCISTRONIC e. How do Eukaryotes find the Initiator AUG? i. Small ribosomal subunit (40S) binds to cap and finds nearest AUG ii. Eukaryotes do not have multiple proteins per mRNA MONOCISTRONIC f. Monocistronic Eukaryotic mRNAs: i. Correct identification of the initiator codon determines the READING FRAME