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Transcript
Chapter 33 Biochemistry 432/832 Protein Synthesis and Degradation December 05 Announcements Protein Synthesis and Degradation Central dogma of biochemistry: DNA --> RNA --> protein Transcription Translation Outline 33.1 Ribosome Structure and Assembly 33.2 Mechanics of Protein Synthesis 33.3 Protein Synthesis in Eukaryotes 33.4 Inhibitors of Protein Synthesis 33.5 Protein Folding 33.6 Post-Translational Processing of Proteins 33.7 Protein Degradation Proof that polypeptides (hemoglobin) grow by addition of new amino acid residues to the carboxyl end Direction of chain growth 0 min unlabeled 4 min 7 min 16 min labeled N-terminus 60 min C-terminus Dintzis experiment, 1961, rabbit reticulocytes labeled with leucine Ribosomes Ribonucleoprotein particles Found in the cytosol, mitochondria and chloroplasts of all cells Move along mRNAs and synthesize proteins Bind and orient mRNAs and aminoacyl-tRNAs Organize interactions between codons and anticodons in aminoacyl-tRNAs Catalyze the formation of peptide bonds between adjacent amino acid residues Ribosome Structure and Assembly E. coli ribosome is 25 nm diameter, 2520 kDa in mass, and consists of two unequal subunits that dissociate at < 1mM Mg2+ 30S subunit is 930 kDa with 21 proteins and a 16S rRNA 50S subunit is 1590 kDa with 31 proteins and two rRNAs: 23S rRNA and 5S rRNA These ribosomes and others are roughly 2/3 RNA 20,000 ribosomes in a cell, 20% of cell's mass Organization of E.coli ribosomes • • Sedimentation coefficient Mass (kDa) Major RNAs Minor RNAs RNA mass RNA proportion Proteins Protein mass Protein Ribosome Small subunit Large subunit 70S 30S 50S 2520 930 1590 16S (1542 b) 23S (2904 b) 5S (120 b) 560 1104 60% 70% 21 31 370 487 40% 30% 1664 66% 857 34% Ribosomal RNA operons in Escherichia coli. Precursor RNA is cleaved to generate 23S, 16S and 5S rRNA as well as tRNA The 16S rRNA fits within the 30S ribosomal subunit The rest of space (peripheral) is occupied by proteins Ribosomal Proteins One of each per ribosome, except L7/L12 (same proteins that differ at N-terminus) with 4 L7/L12 identical except for extent of acetylation at N-terminus Four L7/L12 plus L10 makes "L8" Only one protein is common to large and small subunits: S20 = L26 No similarity (Lys, Arg-rich). The largest is S1 (557 aa) , the smallest is L34 (46 aa) Overall fold of proteins was established less than a year ago Ribosome Assembly/Structure If individual proteins and rRNAs are mixed, functional ribosomes will assemble Structures of large and small subunits have been determined in 2000/2001 A tunnel runs through the large subunit Growing peptide chain is thought to thread through the tunnel during protein synthesis A 3D model for the E.coli ribosome Two views 30S 70S 50S Comparison of ribosomes and tRNAs (two tRNAs may be bound to a ribosome) E.coli ribosome Image reconstruction is based on cryoelectron microscopy Eukaryotic Ribosomes Mitochondrial and chloroplast ribosomes are quite similar to prokaryotic ribosomes, reflecting their supposed prokaryotic origin Cytoplasmic ribosomes are larger and more complex, but many of the structural and functional properties are similar Complexity and size of ribosomes are increased from prokaryotes to lower eukaryotes to higher eukaryotes Conservation of overall RNA structure as well as specific segments of primary sequences Properties of Eukaryotic Ribosomes • • Ribosome Small subunit Large subunit Sedimentation coefficient Mass (kDa) Major RNAs Minor RNAs 80S 40S 60S 4220 RNA mass RNA proportion Proteins Protein mass Protein 2520 60% 1400 2820 18S (1874 b) 28S (4718 b) 5.8S (160 b) 5S (120 b) 700 1820 50% 65% 33 49 700 1000 50% 35% 1700 40% Mechanics of Protein Synthesis All protein synthesis involves three phases: initiation, elongation, termination Initiation involves binding of mRNA and initiator aminoacyl-tRNA to a small subunit, followed by binding of a large subunit Elongation: synthesis of all peptide bonds - with tRNAs bound to acceptor (A) and peptidyl (P) sites. Termination occurs when "stop codon" reached Two binding sites Transfer of a polypeptide to the amino group of amino acid carried by the tRNA in the A site Basic steps in protein synthesis Aminoacyl tRNA is at the A site; polypeptidyl-tRNA is at the P site One codon translocation; tRNA expulsion Location of tRNA binding sites in a ribosome Prokaryotic Initiation The initiator tRNA is one with a formylated methionine: f-Met-tRNAfMet It is only used for initiation, and regular MettRNAmMet is used instead for Met addition N-formyl methionine is first aa of all E.coli proteins, but this is cleaved in about half A formyl transferase adds the formyl group Structure of N-formylmethionyltRNA[Met] Differences with other tRNAs More Initiation Correct registration of mRNA on ribosome requires alignment of a pyrimidine-rich sequence on 3'-end of 16S RNA with a purine-rich part of 5'-end of mRNA The purine-rich segment - the ribosome-binding site - is known as the Shine-Dalgarno sequence Initiation factor proteins, GTP, N-formyl-MettRNAfMet, mRNA and 30S ribosome form the 30S initiation complex Shine-Dalgarno sequences recognized by E.coli ribosomes Events of Initiation 30S subunit with IF-1 and IF-3 binds mRNA, IF-2, GTP and f-Met-tRNAfMet IF-2 delivers the initiator tRNA in a GTPdependent process Loss of the initiation factors leads to binding of 50S subunit The "acceptor site" is now poised to accept an incoming aminoacyl-tRNA Peptide chain initiation 30S subunit (IF-3:IF1) binds mRNA IF-2 delivers the initiator f-Met-tRNA to the P site IF-2 dissociates from 30S subunit GTP hydrolysis is accompanied by IFs release and binding of the 50S subunit A structure of non-hydrolyzable analog of GTP Allowed separation of GTP binding from GTP hydrolysis The Elongation Cycle The elongation factors are vital to cell function, so they are present in significant quantities (EF-Tu is 5% of total protein in E. coli ) EF-Tu binds aminoacyl-tRNA and GTP Aminoacyl-tRNA binds to A site of ribosome as a complex with 2EF-Tu and 2GTP GTP is then hydrolyzed and EF-Tu:GDP complexes dissociate EF-T recycles EF-Tu by exchanging GTP for GDP Elongation factors Factor Mass EF-Tu EF-Ts 43 kDa 74 kDa EF-G 77 kDa Molecules/Cell Function 70,000 Binds tRNA-GTP 10,000 Displaces GDP from EF-Tu 20,000 Binds GTP, promotes translocation of ribosome Peptide chain elongation Reaction of the tRNA-linked peptidyl chain with the a-amino group of an adjacent aminoacyl-tRNA no energy is required for activation Peptidyl Transferase This is the central reaction of protein synthesis 23S rRNA is the peptidyl transferase! The "reaction center" of 23S rRNA - the catalytic bases are among the most highly conserved in all of biology. Translocation of peptidyl-tRNA from the A site to the P site follows Ribosome is a ribozyme (catalytic rRNA) Catalytic center is located in the 50S particle The peptidyl transferase center of 23S rRNA Ribosome is ribozyme Puglisi JD, Blanchard SC, Green R, Nat Struct Biol 2000 Oct;7(10):855 Approaching translation at atomic resolution. Atomic resolution structures of 50S and 30S ribosomal particles have recently been solved by X-ray diffraction In the 50S structure, the active site for peptide bond formation was localized and found to consist of RNA. The ribosome is thus a ribozyme In the 30S structure, tRNA binding sites were located The 30S subunit particle has three globular domains, and relative movements of these domains may be required for translocation of the ribosome during protein synthesis Ribosome structure Yusupov MM, Yusupova GZ, Baucom A, Lieberman K, Earnest TN, Cate JH, Noller HF. Science 2001 Mar 29 Crystal Structure of the Ribosome at 5.5 A Resolution. We describe the crystal structure of the complete Thermus thermophilus 70S ribosome containing bound mRNA and tRNAs at 5.5 A resolution. All of the 16S, 23S and 5S rRNA chains, the A-, P- and E-site tRNAs, and most of the ribosomal proteins can be fitted to the electron density map. The core of the interface between the 30S small subunit and the 50S large subunit, where the tRNA substrates are bound, is dominated by RNA, with proteins located mainly at the periphery, consistent with ribosomal function being based on rRNA. In each of the three tRNA binding sites, the ribosome contacts all of the major elements of tRNA, providing an explanation for the conservation of tRNA structure. The tRNAs are closely juxtaposed with the intersubunit bridges, in a way that suggests coupling of the 20 to 50 A movements associated with tRNA translocation with intersubunit movement. The Role of GTP Hydrolysis Two GTPs are hydrolyzed for each amino acid incorporated into peptide. Hydrolysis drives essential conformation changes Total of four high-energy phosphate bonds are expended per amino acid residue added - three GTP here and two in amino acid activation via aminoacyl-tRNA synthesis Movement of tRNAs during translation Relative positions of tRNA molecules in a ribosome during peptidyl transfer and translocation Peptide Chain Termination Proteins known as "release factors" recognize the stop codon at the A site Presence of release factors with a nonsense codon at A site transforms the peptidyl transferase into a hydrolase, which cleaves the peptidyl chain from the tRNA carrier Termination of protein synthesis