Translation and Proteins Translation Protein synthesis In the late 1950s Paul Zamecnik and Mahlon Hoagland noticed that the RNA in particular cytoplasmic fraction became labeled with 14C amino acids and that the labeled RNA was subsequently able to transfer the amino acids to microsomal protein rough ER Conclusion the RNA later named transfer RNA of tRNA functions as an intermediate carrier of amino acids in protein synthesis Zamecnik and Hoaglandlaater contributed to our understanding of how transfer process works Translation depends on mRNA rRNA and tRNA Each codon triplet specifies one amino acid To connect two languages Francis Crick proposed the adaptor hypothesis in 1955 wherein transfer RNA tRNA adapts each codon triplet in mRNA to the correct amino acid Anticodon in tRNA pairs with codon of mRNA Site of protein manufacture is the ribosome Ribosome is most intricate of cell structures about 10 000 ribosomes bacterial cell ribosomes are made of both RNA and protein differ between prokaryotes and eukaryotes Summary of differences between Prokaryotic and Eukaryotic Ribosomes Prokaryotes 50 S subunit 23 S rRNA 2904 n 31 proteins 5 S rRNA 120 n 16S rRNA 1541 n 21 proteins 30 S subunit in E coli Eukaryotes One transcript produces 23S 16S and 5S rRNA 7 copies of gene 60 subunit 28 S rRNA 4718 n 49 proteins 5 S rRNA 120 n 5 8 s rRNA 160 n 18 S rRNA 1874 n 33 proteins 40 S subunit One transcript produces 28S 18S and 5 8 s rRNA 120 copies of Transfer RNA tRNA gene in fly Small about 83 nucleotides stable RNA molecules Cleaved from larger percurser RNA Contain several modified nucleotides which are modified AFTER transcription figure below Cloverleaf model of tRNA Anticodon loop contains anticodon sequence The 3 end pCpCpA is where amino acid is covalently bound 5 Gp at end of every tRNA 3 D model of tRNA Shape of acceptor stem allows recognition by charging enzymes Charging tRNA loading the gun Aminoacyl tRNA synthetases charge a tRNA so it is covalently linked to its cognate amino acid Could be 61 tRNAs but so far only about 32 know other codons can be reorganized by same tRNA due to wobble at 3rd position Only about 20 synthetases needed in a given cell Synthetase amino acid ATP form an aminoacyl adenylic acid intermediate This complex reacts with isoaccepting tRNAs to bond the amino acid to 3 end of tRNA and release of AMP Translation of mRNA can be divided into 3 steps Initiation Elongation and Termination a continuous dynamic process small subunit mRNA charged initiator tRNA GTP Mg 3 protein initiation factors form initiation complex Initiator tRNA AUG requires formylmethione fmet in bacteria methionine in eukaryotes Complex binds to Shine Dalgarno only purines i e AGGAGG sequence 5 of AUG codon fmet tRNA then binds setting frame of translation Translation Elongation When two subunits are combined then three sites exist P site peptidyl charged initiator binds here A site aminoacyl codon determines next charged tRNA E site exit area for exiting used tRNA Peptide transferase catalyzes formation of peptide bond between two amino acids on tRNAs tRNA amino acid bonds in P site is broken and tRNA released via e site Entire mRNA tRNA aa1 aa2 complex shifts 3 nucleotides to tRNA peptide in P site and tRNA amino acid in A site 15aa sec at 37 degrees C in E coli error rate is 1 in 10 000 Binding of EF Tu and tRNAs to the ribosome seen by cryo EM A Aminoacyl tRNA in A T site in ternary complex peptidyl tRNA in P site and deacylated tRNA in Esite B Pre translocation ribosome with peptidyl tRNA in A site deacylated tRNAs in P and E site Translation Termination When ribosome complex encounters a stop codon in A site then GTP dependent release factors cleave polypeptide from last tRNA tRNA is released and also the polypeptide Many ribosomes can consecutively translate a mRNA can start before the previous ribosomes is finished leading to formation of polyribosomes In eukaryotes larger ribosomes mRNAs are long lived hrs rather than min need 5 cap on mRN Kozak sequence replaces Shine Dalgarno sequence tRNAimet is used more translation factors many ribosomes associated with ER secret polypeptides Polyribosomes Figure 13 9 polyribosomes visualized by electron microscopy In prokaryotes transcription and translation are coupled since there is not nuclear membrane ribosomes can be seen binding to mRNA 13 9a and proteins emerging from there in 13 9b Efficient use of protein components High Resolution Studies Reveal Details About the Functional Prokaryotic Ribosome V Ramakrishnan and H Noller generated and examined crystals of large ribosomal subunits Structures confirm that RNA is the major catalytic player in ribosome function with proteins being on the periphery tRNA directly contacts the rRNA Details support the wobble hypothesis proposed by Francis Crick Translation is more complex in Eukaryotes In eukaryotes the ribosomes are larger with more proteins attached mRNAs are longer lived in eukaryotes Transcription and translation are uncoupled in eukaryotes with more layers of regulation The 7mG cap on mRNA is essential for efficient translation Kozak sequence functions like Shine Dalgarno sequence in prokaryotes A unique tRNA tRNAI Proteins are secreted through a tunnel into the endoplasmic reticulum met is used for translation initiation One Gene one Enzyme Hypothesis Beadle and Tatum induced nutritional auxotrophic mutants in Neurospora Could correct deficiency by supplementing with appropriate biochemical Genes and Enzymes Analysis of Biochemical Pathways By analyzing which chemical overcomes the block in each particular mutant then the pathway could be determined Precursor Ornithine Citrulline Arginine They demonstrated that genetics and biochemistry had much in common a mutation could be found nearly any enzyme activity Studies of Human Hemoglobin Established that One Gene encodes One Polypeptide Not all proteins are enzymes some are structural as opposed to metabolic one gene one proteins is more appropriate Some proteins have many subunits one gene one polypeptide is most appropriate Sickle Cell Anemia Molecular Disease 1949 Neel and Beat demonstrated this is a Mendelian trait Blood cells sickle under low oxygen conditions Heterozygotes have sickle cell trait have much less sickling Studied altered migration of hemoglobin proteins fragments found single amino acid changed Valine instead of Glutamic acid in 6 position of beta change Analyze hemoglobin from normal and sickle cell patients to