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5/2/16 Protein Synthesis II Chapter 27 962 – 968 (independent study) 968– xxx Ribosomes – E. coli vs Eukaryo4c • Ribosomes are a mixture of proteins and one or more rRNA molecules. • Job of ribosome is to create the environment to allow tRNA and mRNA to bind and to catalyze pepLde backbone formaLon • Both have two subunits whose structures are sLll only parLally solved but salient features are known (more later) • Significant differences between organisms. • Important to understand the overall composiLon and compare between the two… Secondary structure of E. coli rRNA 1 5/2/16 Key features of Ribosomes • Sub-‐unit interface is mostly rRNA, with the various proteins found more located in the back side. tRNA / mRNA interface is primarily rRNA with protein mostly on the surface Key features of Ribosomes • Three sites P (pepLdyl) A (amino acyl-‐tRNA) and E (exit) • mRNA binds to interface via rRNA secondary structure – Note sharp bend of mRNA at E site – more later • Large subunit – pepLdyl transferase center • Small subunit – decoding center Ribosomal Proteins • Most are globular with some sort of tail. • Tail (usually basic side chains) interact at interface of subunits through salt bridges via RNA negaLve charged phosphate groups • Supports the RNA world’s idea that proteins were adapted to be used in support of RNA • ff Three sites to ribosome • Ribosome binds all three tRNA in same manner, but the mRNA is distorted at E site. • PepLde exits into tunnel on large subunit. -‐ folding occurs aZer exiLng due to size constricLon 2 5/2/16 Basic Reac4on – pep4de elonga4on • New amino acid’s N nucleophilcally a\acks the pepLdyl-‐tRNA ester in the P site, moving the pepLde to the A site. Note that the growing pepLde chain is TRANSERED to the aa-‐ tRNA, not the other way around! Nucleophilic a\ack happens at the same Lme as a proton transfer from amino acid and 2’OH of P site ribose Remember that the aminoacyl-‐tRNA ester bond has nearly the same dG as ATP thus addiLonal energy is not required Ribosome’s acLve site is in the large subunit and the nearest protein is too far to be involved, thus this is an RNA catalyzed (ribozyme) reacLon!!! ReacLon is catalyzed by removal of H from amino acid N group by N3 adenine (invariant bp). The protonated N of RNA adenine also stabilizes the intermediate state Ini4a4on – bact vs eukaryote • Starts with Met-‐tRNA – N-‐formyl (fMet) in bacteria – fMet is a different tRNA than Met-‐ tRNA but both bind same codon (AUG) thus the ribosome and iniLaLng proteins must recognize the tRNA differences to avoid fMet being incorporated to growing strand… • What is the consequence of fMet in elongaLon? Ini4a4on Requires Addi4onal Non-‐Ribosomal Proteins 3 5/2/16 Eukaryote Ini4a4on • Up to 12 different iniLaLon proteins (eIF#) • Overall similar to bacterial iniLaLon. But no Shine-‐ Dalgarno… • Eukaryote mRNA has a m7 G cap 5’ cap and a polyA 3’ end used in recogniLon by iniLaLng factors – Form a pre-‐iniLaLon complex. – Met-‐tRNA (not fMet – that sets off human immune system – warning of bacterial infecLon) 4 5/2/16 5
