Camerini 4 - Lectures 4 Initiation elongation and...

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1 Lectures 4: Initiation, elongation and termination of protein synthesis We will discuss the following: The structure of ribosomes Sequential polypeptide synthesis from the N to C terminus Ribosomes read mRNA in the 5’->3’ direction tRNA binding sites (A, P and E) in the ribosome Initiation Elongation Termination
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2 Structure of prokaryotic 50S large ribosomal subunit Note the core of this (we are looking up into it, as though we were just 30S subunits). This is the active site, where the important business of the ribosome takes place (e.g. peptide bond formation). The 30S subunit contains the decoder. Proteins (L1, etc) and rRNAs (5S, 23S)
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3 Cryo-electron micrographic data on the overall structure of the 70S ribosome 30S (yellow) landmarks: -ch = Channel for mRNA 50S (blue) landmarks: -CP=central protuberance -T=tunnel (protein extrusion site?) -E1, E2: branch points in tunnel thought be be exit point of extruded polypeptide chain Also note sites A and P in the structure. We will talk about these in detail, since they are where the “action” takes place.
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4 The structure of the 23S rRNA (grey) from Haloarcula marismortui . Ribosomal proteins are shown in yellow. Catalysis occurs in the center of this structure (denoted by the star). This perspective is that from the 30S subunit. 23S rRNA Of note: not much protein in the center where catalysis takes place. Think ribozymes!
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5 How are proteins synthesized? Scientists knew that proteins were linear heteropolymers, but in the early 1960’s, they did not know how they were synthesized. They did know that genes and proteins are colinear and that ribosomes make proteins. But, they didn’t know how the ribosomes did this. For example, was the synthesis sequential, or was there random addition of AAs onto a fixed template. If it was sequential, did AAs get added to the amino or carboxy terminus? To address these questions, Nirenberg used a variation on his previous methods.
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6 Ribosomes read mRNA in the 5’ -> 3’ direction Nirenberg: synthesized artificial mRNA, cell-free system. The genetic code was already known. The artificial RNA was polyA, but had one C at the 3’ end (he’d gotten very good at making artificial RNAs by this point). Then, they determined which AAs were incorporated into the protein synthesized: Nucleotide sequence Protein Sequence Direction of Synthesis -AAAAAA(AAA)nAAC-3Õ Lys-Lys-(Lys)n-Asn ->3Õ -CAAAAA(AAA)nAAA-5Õ Gln-Lys-(Lys)n-Lys -5Õ Since he got proteins with a bunch of poly-lysine followed by an asparigine, he proved that mRNA is read in the 5’->3’ direction.
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7 What we know so far …..
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