Translation Translation is the process of converting an RNA sequence into a protein. The Genetic Code how to code for 20 amino acids with only four different bases? a triplet code would allow 64 different amino acids Tsugita and Frankel-Conrat used point mutations in TMV to show that the code was nonoverlapping Crick used acridine mutagens (proflavin) to create deletions and insertions no gaps in the code a specific start point as three insertions or deletions could produce wild-type activity -the code must be triplet Nirenberg and Matthaei - in vitro translation of poly U = polyphenylalanine Khorana - used poly(UC) to show that the code has an odd number of bases (he got poly(serine-leucine)) a repeating trinucleotide gave three different homopolypeptides a repeating tetranucleotide gave a repeating tetrapeptide here's a more complete explanation of the Nirenberg and Khorana experiments, from DNA from the Beginning Try to answer this question about a dinucleotide at the Biology Project Nirenberg showed that a trinucleotide could bind an aminoacyl-tRNA in vitro - he and Khorana then broke the code the code is degenerate there is more than one codon for an amino acid (up to six different codons for some amino acids) except for methionine and tryptophan there are special start and stop codons AUG codes for methionine and is also the start codon UAA, UAG and UGA don't code for amino acids and are the stop codons (a special protein, releasing factor, binds to them and stops translation) The complete sequence of the phage MS2 and of the proteins coded for by the MS2 genes confimed the genetic code The code is almost universal, with only a few minor variations, particulary in mitochondria tRNA tRNA is the translator aminoacyl synthetases puts amino acids on one end of the tRNA if the amino acid is changed after it is linked to the tRNA the new amino acid will be inserted as if it were the original amino acid cysteine-tRNACys treated with nickel hydride gives alanine-tRNACys proteins made with this tRNA have alanine where cysteine should be each tRNA has an anticodon that can base pair with the codon code is degenerate Crick's wobble hypothesis - the first base base of the anticodon can form unusual base pairs (G to U for instance), also unusual bases such as inosine can pair with more than one nucleotide some amino acids have more than one tRNA only about 32 different tRNA's the aminoacyl synthetases recognize various characteristics of the tRNA molecule three codons have no tRNA - the stop codons the stop codons are recognized by protein releasing factors that terminate translation mutations that create premature stops can be suppressed by a mutation in a tRNA that creates a new tRNA that can recognize the stop codon UAG = amber, UGA = opal, UAA = ochre Try and answer this question about translation at the Biology Project Translation Ribosomes - the site of protein synthesis two subparticles - 30S and 50S in prokaryotes, 40S and 60S in eukaryotes 30S = 16SrRNA + 21 proteins 50S = 23SrRNA + 5S rRNA + 34 proteins 30S + 50S gives a 70S ribosome; in eukaryotes 40S + 60S gives an 80S ribosome self-assembly multiple ribosomes can traslate from one mRNA at the same time (polysomes) initiation initiation factors + 30S + mRNA + GTP + fMet-tRNA requires ribosome binding sites on the mRNA (Shine-Dalgarno sequence 5'-AGGAGGU-3' an AUG (sometimes GUG) downstream of the Shine-Dalgarno sequence is the first codon used elongation mRNA is read 5' to 3' aminoacyl tRNA binds at the A site of the ribosome peptidyl transferase makes a peptide bond between the amino acid at the P site and the one in the A site, releasing the peptide from the P site the ribosome translocates 3 nucleotides moving the peptide chain to the P site using one GTP for energy termination a protein release factor recognizes a stop codon in the A site and releases the peptide chain from the tRNA at the P site Variations in Transcription and Translation in Prokaryotes and Eukaryotes Eukaryotic mRNA is heavily processed, unlike prokaryotic mRNA the initial transcript is called hnRNA (heterogenous RNA) because it was originally this unknown RNA found with a large range of sizes only in the nucleus the hnRNa is has sequences removed from each end and then a 5' cap (methyl guanasine added 5' to 5') and a poly A tail (a string of about 300 As added to the 3' end of the RNA) are added introns are then spliced out - various stretches of RNA sequence are cut out of the transcript and then the remaining exons are spliced back together, the introns are presumabely degraded the mature mRNA is then exprted to the cytoplasm for translation Here is a more complete description of the discovery of introns, from the "DNA from the Beginning" site Try answering these quesions about eukaryotic transcription from the Biology Project: question1, question2, question3, question4, question5, question6, question7 PROCARYOTIC EUCARYOTIC Promoter Pribnow's Box TATAATG TATA Box TATAAATA RNA Polymerase One Type Three Types Pol I - rRNA Pol II - mRNA Pol III - tRNA Product of Transcription mRNA, no cap or tail hnRNA which is modified with Methyl Guanosine Cap & Poly A Tail Introns Very rare Very common Translation Starts while the mRNA is still being synthesized (no nucleus) Only in the cytoplasm on processed mRNAs Ribosomes 70S 80S Start Codon AUG (f-met) AUG (met) Initiation Sequences Shine-Delgarno sequences Weak consensus sequence mRNA Polycistronic possible Monocistronic need cap for initiation Origin of Life Conserved code argues strongly for one origin DNA replication, transcription and translation are too complicated Current hypothesis: an RNA origin RNA is present and required for replication (the primer, telomeres), transcription, and translation (mRNA, rRNA and tRNA) RNA can both code for information (RNA viruses) and act enzymatically (self-splicing, peptidyl transferase, ribozymes, etc.) For more on the origina of life chck out this animation of the Urey-Miller experiments and Cech's discovery of ribozymes, from "DNA from the Beginning" Try this question from the Biology Project This document is maintained by: Jeff Bell Last Update: Friday, March 1, 2002