Lecture March 11 CvR 1 Transition Mechanisms Turning nucleotides into properties of life o Transcription Makes RNA by making nucleotides from purines and pyrimidines Starting with pre mRNA splicing occurs as a form of regulation of mRNA Followed by nuclear export o Regulation steps occur here as well After nuclear export o Translation most common result o IRES mediated translation different type of mRNA function o mRNA decay can influence gene expression Time for decay is related to amount of expression produced per mRNA o mRNA localization Some go to produce proteins in very specific cellular locations it s not just the protein products that are localized Central Dogma Classical o DNA DNA Replication o DNA RNA Transcription o RNA protein Translation o DNA protein done in vitro However the opposite can occur o RNA DNA reverse transcription o RNA RNA ribozyme function Converting between languages DNA nucleotide language RNA nucleotide language Protein amino acid language Why different languages Why not others Complicated language being decoded back and forth o Protein RNA DNA protein cannot be done o 20aa gives rise to more possibilities than 4 nucleotides o More diversity with different side chains o Possible variety of just 100aa proteins 20100 vs 4100 Amino acids give WAY more diversity o 20100 aa is more things than in the universe What tool helps convert the two languages of nucleotides and amino acids o tRNA tRNA Possess a D Loop and T Loop along the outside of the clover structure left and right side respectively o Have modified nucleotides in them Anticodon loop at the bottom o Also has modified nucleotides o Reads the mRNA therefore does the conversion o MOST IMPORTANT PART o Possesses the anticodon loop 43 codon in triplet 4 nucleotides Mathematically 64 codons 64 tRNA No 3 for stop codons 61tRNA No there s the initiator methionine codon 61 tRNA Nope Why Wobble base pairing o Also has a CCA amino acid acceptor group amino acid attachment site Aminoacyl tRNA synthetases Conjugated amino acids bound to tRNA done by this enzyme 20 available one for each amino acid o Makes amino acyl bond to tRNA Combines ATP with amino acid to make ATP amino acyl intermediate o Amino acid adenine ribose Mechanism o Amino acid binds so does ATP loss of pyrophosphate probably irreversible o tRNA comes in reacts with the transition state amino acyl AMP o Produces Aminoacyl tRNA a charged group that leaves How do the 20 aminoacyl tRNA synthetases differ from each other o Probably in their anticodon binding domain o Definitely binding site to tRNA and amino acid binding site Group right before CCA amino acid binding site is different for each tRNA o tRNAs differ in the region before their CCA attachment site and in the anticodon loop Nucleotides are monstrous in relation to proteins Wobble Base Pairing The third nucleotide in the anticodon loop can recognize other things when reading 5 3 on the mRNA o G should bind C but can also bind U G C U U A G I A C U o Back in the codon table almost every amino acid reacts with several codons from wobble base pairing o Need less tRNAs to help juggle all the codons Unusual Amino Acids If you want to take up modified amino acids Reengineer the tRNA synthetase recognize new amino acids o To recognize the amino acid o To recognize the tRNA the protein just once o Amber stop codon Modify the tRNA anticodon loop to incorporate modified amino acids to ta specific spot in You can make a tRNA for the amber stop codon and modified tRNA will add the amino acid when transcription stops o 4 base codon Larger codon to make it insert into the protein Would have to be careful to not mess up the frame Make sure it s not inserted anywhere else Modify the codon The Ribosomes Why are enzymes like aminoacyl tRNA synthetases or ribosomes o They speed up the rate of reaction like crazy so organic process can occur naturally Ribosomes aren t the most important thing in translation o They re just a catalyst Ribonucleoprotein of 2 subunits o Ribozymes RNA catalyst Exist in prokaryotes and eukaryotes o Differ in their size speed and composition Eukaryotic ribosomes function at 6 9aa sec o Prokaryotes work 3x faster at 17 21aa sec Ribosomes comparison Prok Vs Euk Prokaryote is much faster than euaryote Size is larger in eukaryotes o 2 3MDa vs 4 3MDa Composition o Prokaryote 3rRNA 2 large subunits 50S 30S o Eukaryotes 4rRNA 3 large subunit rRNA 60S 40S Complexity o Eukaryotes are made of more proteins more sites of regulation and control rRNA has primary secondary and tertiary structure o Can have plenty of stem loop structures etc Where are they produced o Made in the nucleolus then exported out o Proteins for ribosome made in the cytosol o 5S rRNA comes from nucleus not nucleolus Template of translation Cap 5 UTR Open reading frame coding sequence 3 UTR polyA Tail Biology of an mRNA o Eukaryotic Prokaryotic mRNA o Don t have 5 cap o Don t necessarily have 3 polyA tail o Usually polycistronic differing largely from ours UTR 5 and 3 UTR influence localization stabilization and translation of mRNAs They can bind various protein and miRNA factors o Increase and decrease transcription and degradation o Mostly binding in the 3 UTR to stabilize the message mRNA degradation is a good thing certain mRNA need to die soon 7 methylguanylate cap 5 cap or m7G Added co transcriptionally Involved in splicing Enables nuclear export o No cap no export from nucleus Involved in recognition of mRNA for translation Stabilizes mRNA from decay Cap is 5 5 link by a methylated guanine Additional methylation can also occur on the 2 hydroxyls of ribose groups o Not all just the first 1 or 2 of the nucleotides right after the cap snRNAs get a m2 2 7G methylation pattern o specific methylation patterns can differentiate between different types of mRNA Mechanism of addition of 5 cap At the 5 end you have a triphosphate from the final added nucleotide Enzyme comes to remove 1 phosphate GTP comes in and is added 5 5 triphosphate link with loss of its pyrophosphate o Protects mRNA from degradation because normally degradation enzymes target the 5 3 mRNA Use SAM as methyl donator to give methyl group to guanine on C7 Optional add more SAM to methylate the 2 OH of the first few nucleotides in the mRNA This is all Cap 1 o Then exported from the nucleus In higher eukaryotes you ll add the 2nd Cap methylated 2 OH after export To watch this you can add radioactive GTP and see addition of Guanine cap Capping process