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The Ribosome, rRNA and mRNA (3.1) Lecture 3 The Ribosome, rRNA and mRNA Major players in protein synthesis: mRNA, tRNA and the ribosome mRNA Messenger RNA, a copy of DNA blueprint of the gene to be expressed. tRNA Aminoacyl transfer RNA, also called anticodon or adaptor molecule. One or more tRNAs for each amino acid. Supply Ribosome A very large complex of several rRNAs (ribosomal RNA) and many protein molecules. Total molecular weight almost 3 million dalton. Factory Protein Polypeptide chain with sequence dictated by the mRNA sequence. Also called the gene product. Product The Ribosome http://bass.bio.uci.edu/~hudel/bs99a/lecture22/index.html (1 of 4)5/25/2007 9:34:42 AM Information The Ribosome, rRNA and mRNA (3.1) Ribosomes can be found either free in the cytosol (cytoplasm) or attached to intracellular membranes. Free ribosomes ● ● ● ● Found in the cytosol. May occur as a single ribosome or in groups known as polyribosomes or polysomes. Occur in greater number than bound ribosomes in cells that retain most of their manufactured protein. Responsible for proteins that go into solution in the cytoplasm or form important cytoplasmic structural or motile elements. http://bass.bio.uci.edu/~hudel/bs99a/lecture22/index.html (2 of 4)5/25/2007 9:34:42 AM The Ribosome, rRNA and mRNA (3.1) Bound ribosomes ● ● ● Found bound to the exterior of the endoplasmic reticulum (ER) constituting the rough ER. Occur in greater number than free ribosomes in cells that secrete their manufactured proteins (e.g., pancreatic cells, producers of digestive enzymes). Responsible for proteins that insert into membranes or are packaged into vesicles for storage in the cytoplasm or export to the cell exterior. Electronmicrograph of ribosomes (black dots) attached to the rough endoplasmic reticulum. ● ● ● ● ● ● About 15,000 ribosomes in a single E. coli cell, comprising ~25% of the dry cell mass. All ribosomes within one cell are identical. All ribosomes are composed of two subunits (called small and large). A substantial fraction of ribosomes are dissociated into free subunits in the cell. Prokaryotic and eukaryotic ribosomes differ in composition. Mitochondrial ribosomes resemble prokaryotic ribosomes. Next: The Composition of Ribosomes http://bass.bio.uci.edu/~hudel/bs99a/lecture22/index.html (3 of 4)5/25/2007 9:34:42 AM The Ribosome, rRNA and mRNA (3.1) Please report typos, errors etc. by EMAIL (mention the title of this page). http://bass.bio.uci.edu/~hudel/bs99a/lecture22/index.html (4 of 4)5/25/2007 9:34:42 AM The Composition of Ribosomes (3.2) The Composition of Ribosomes Prokaryotic ribosomes ● Sedimentation coefficient: 70S ❍ ❍ small subunit: 30S ■ One rRNA molecule (16S) ■ 21 different proteins, designated S1-S21 large subunit: 50S ■ Two rRNA molecules (5S and 23S) ■ 31 different proteins, designated L1-L31 (L12 is present in four copies) http://bass.bio.uci.edu/~hudel/bs99a/lecture22/lecture3_2.html (1 of 4)5/25/2007 9:34:44 AM The Composition of Ribosomes (3.2) The sedimentation coefficient is measured in Svedberg units (S): the rate of sedimentation of a component in a centrifuge is related both to the molecular weight and the 3-D shape of the component. The rRNA components of the prokaryotic ribosome Type Approximate number of nucleotides Subunit location 16S 1,542 30S 5S 120 50S 23S 2,904 50S RNA function and turnover in the cell Nucleotides % of Synthesis % of Total RNA Thousands 500-6000 40-50 3 3 (23S, 16S, 5S) 2904, 1542, 120 50 90 Stable ~50 73-93 3 7 Stable Different Kinds mRNA Messenger rRNA Ribosome Type tRNA Function Adapter http://bass.bio.uci.edu/~hudel/bs99a/lecture22/lecture3_2.html (2 of 4)5/25/2007 9:34:44 AM Stability T1/2 = 1-3 min The Composition of Ribosomes (3.2) Separation of ribosomal proteins by 2D gel electrophoresis (a) small (30S) subunit; (b) large (50S) subunit http://bass.bio.uci.edu/~hudel/bs99a/lecture22/lecture3_2.html (3 of 4)5/25/2007 9:34:44 AM The Composition of Ribosomes (3.2) In 1968, Dr. M. Nomura, a professor here at UCI, was the first to show that the 30S subunit could be reassembled from the individual components. He found that the order of addition of components was critical. Eukaryotic ribosomes ● Sedimentation coefficient: 80S ❍ ❍ small subunit: 40S ■ One rRNA molecule (18S) ■ 33 different proteins, designated S1-S33 large subunit: 60S ■ Three rRNA molecules (5S, 5.8S, and 28S) ■ 50 different proteins, designated L1-L50 The rRNA components of the eukaryotic ribosome Type Approximate number of nucleotides Subunit location 18S 1,900 40S 5S 120 60S 5.8S 156 60S 4,700 60S 28S Next: Simplified Overview of Translation Please report typos, errors etc. by EMAIL (mention the title of this page). http://bass.bio.uci.edu/~hudel/bs99a/lecture22/lecture3_2.html (4 of 4)5/25/2007 9:34:44 AM Simplified Overview of Translation (3.3) Simplified Overview of Translation 1. Formation of the initiation complex 2. Elongation of the polypeptide chain (one repetition of the steps a, b and c for every amino acid incorporated into the protein being synthesized): a: binding of the aminoacyl-tRNA b: peptide bond formation c: translocation 3. Termination Rate of synthesis ● ● Elongation is the rate limiting step in protein synthesis In E. coli at 37 degrees C: http://bass.bio.uci.edu/~hudel/bs99a/lecture22/lecture3_3.html (1 of 2)5/25/2007 9:34:47 AM Simplified Overview of Translation (3.3) ❍ ❍ ❍ ribosome passes through 15 codons per second 300 amino acid polypeptide made in 20 seconds 15,000 ribosomes per cell can make 750 molecules of a 300 amino acid protein per second Next: Structure of the Ribosome Please report typos, errors etc. by EMAIL (mention the title of this page). http://bass.bio.uci.edu/~hudel/bs99a/lecture22/lecture3_3.html (2 of 2)5/25/2007 9:34:47 AM The Structure of the Ribosome (3.4) The Structure of the Ribosome (Prokaryotic ribosomes) Proposed secondary structure of 16S rRNA http://bass.bio.uci.edu/~hudel/bs99a/lecture22/lecture3_4.html (1 of 6)5/25/2007 9:34:47 AM The Structure of the Ribosome (3.4) ● ● Many regions are self-complementary and capable of forming double helical segments Secondary structure is more highly conserved than primary sequence, i.e. complementary mutations evolve to maintain base paring. 3-Dimensional structure of the 70S ribosome http://bass.bio.uci.edu/~hudel/bs99a/lecture22/lecture3_4.html (2 of 6)5/25/2007 9:34:47 AM The Structure of the Ribosome (3.4) The large subunit has a tunnel about 10 nm long and 2.5 nm in diameter. This tunnel is thought to be the channel that newly assembled polypeptide chains pass through on their way out of the ribosome. The two subunits interact very tightly and form the active site http://bass.bio.uci.edu/~hudel/bs99a/lecture22/lecture3_4.html (3 of 6)5/25/2007 9:34:47 AM The Structure of the Ribosome (3.4) Different views: in yellow the 30S (small) subunit; http://bass.bio.uci.edu/~hudel/bs99a/lecture22/lecture3_4.html (4 of 6)5/25/2007 9:34:47 AM in blue the 50S (large) subunit. The Structure of the Ribosome (3.4) Landmarks of the 30S subunit: h, head; p, platform; ch, channel presumed to be the conduit for mRNA; sp, spur. Landmarks of the 50S subunit: CP, central protuberance; St, L7/L12 stalk; L1, L1 protein; IC, interface canyon; T, tunnel presumed to be the conduit for the nascent polypeptide chain; T1 and T2, lower tunnel http://bass.bio.uci.edu/~hudel/bs99a/lecture22/lecture3_4.html (5 of 6)5/25/2007 9:34:47 AM The Structure of the Ribosome (3.4) segments, leading to alternative exit sites E1 and E2, respectively. Next: The X-ray Structure Please report typos, errors etc. by EMAIL (mention the title of this page). http://bass.bio.uci.edu/~hudel/bs99a/lecture22/lecture3_4.html (6 of 6)5/25/2007 9:34:47 AM The X-ray Structure of the Ribosome (3.4b) The X-ray Structure of the Ribosome Two back-to-back landmark papers in the journal Science (August 2000) provided a whole new level of information of what ribosomes look like in detail, and specifically, how the crucial peptide bond formation is catalyzed: The Complete Atomic Structure of the Large Ribosomal Subunit at 2.4 Å Resolution Nenad Ban, Poul Nissen, Jeffrey Hansen, Peter B. Moore, Thomas A. Steitz The large ribosomal subunit catalyzes peptide bond formation and binds initiation, termination, and elongation factors. We have determined the crystal structure of the large ribosomal subunit from Haloarcula marismortui at 2.4 angstrom resolution, and it includes 2833 of the subunit's 3045 nucleotides and 27 of its 31 proteins. The domains of its RNAs all have irregular shapes and fit together in the ribosome like the pieces of a three-dimensional jigsaw puzzle to form a large, monolithic structure. Proteins are abundant everywhere on its surface except in the active site where peptide bond formation occurs and where it contacts the small subunit. Most of the proteins stabilize the structure by interacting with several RNA domains, often using idiosyncratically folded extensions that reach into the subunit's interior. Science (2000) 289, 905-920. The Structural Basis of Ribosome Activity in Peptide Bond Synthesis Poul Nissen, Jeffrey Hansen, Nenad Ban, Peter B. Moore, Thomas A. Steitz Using the atomic structures of the large ribosomal subunit from Haloarcula marismortui and its complexes with two substrate analogs, we establish that the ribosome is a ribozyme and address the catalytic properties of its all-RNA active site. Both substrate analogs are contacted exclusively by conserved ribosomal RNA (rRNA) residues from domain V of 23S rRNA; there are no protein side-chain atoms closer than about 18 angstroms to the peptide bond being synthesized. The mechanism of peptide bond synthesis appears to resemble the reverse of the acylation step in serine proteases, with the base of A2486 (A2451 in Escherichia coli) playing the same general base role as histidine-57 in chymotrypsin. The unusual pKa (where Ka is the acid dissociation constant) required for A2486 to perform this function may derive in part from its hydrogen bonding to G2482 (G2447 in E. coli), which also interacts with a buried phosphate that could stabilize unusual tautomers of these two bases. The polypeptide exit tunnel is largely formed by RNA but has significant contributions from proteins L4, L22, and L39e, and its exit is encircled by proteins L19, L22, L23, L24, L29, and L31e. Science (2000) 289, 920-930. http://bass.bio.uci.edu/~hudel/bs99a/lecture22/lecture3_4b.html (1 of 6)5/25/2007 9:34:54 AM The X-ray Structure of the Ribosome (3.4b) The first paper provides a wealth of structural information on how the components of the large (50S) subunit are arranged: http://bass.bio.uci.edu/~hudel/bs99a/lecture22/lecture3_4b.html (2 of 6)5/25/2007 9:34:54 AM The X-ray Structure of the Ribosome (3.4b) ● ● the rRNA (in gray) is forming the core the ribosomal proteins (yellow) are mostly on the surface It also shows the exit tunnel and suggests that not only an extended polpypeptide would fit through it, but also one in an alpha-helical conformation: http://bass.bio.uci.edu/~hudel/bs99a/lecture22/lecture3_4b.html (3 of 6)5/25/2007 9:34:54 AM The X-ray Structure of the Ribosome (3.4b) The second paper provides a detailed mechanism for peptide bond formation based on the x-ray structure. It http://bass.bio.uci.edu/~hudel/bs99a/lecture22/lecture3_4b.html (4 of 6)5/25/2007 9:34:54 AM The X-ray Structure of the Ribosome (3.4b) highlights the central role of the rRNA, and specifically that of base A2486 (RIBOZYME!): Next: Supplemental Material http://bass.bio.uci.edu/~hudel/bs99a/lecture22/lecture3_4b.html (5 of 6)5/25/2007 9:34:54 AM The X-ray Structure of the Ribosome (3.4b) Please report typos, errors etc. by EMAIL (mention the title of this page). http://bass.bio.uci.edu/~hudel/bs99a/lecture22/lecture3_4b.html (6 of 6)5/25/2007 9:34:54 AM Supplemental Material (3.5) Supplemental Material This part won't be on the final! If you are interested how findings like these are presented in an original research article, you should take a look at the 1995 paper in the journal Nature: "A model of protein synthesis based on cryo-electron microscopy of the E. coli ribosome" by Frank, J., Zhu, J., Penczek, P., Li, Y., Srivastava, S., Verschoor, A., Radermacher, M., Grassucci, R., Lata, R.K. and Agrawal, R.K. Nature 376, 441-444 (1995). And for comparison, check out the more recent back-to-back X-ray papers in the journal Science: "The Complete Atomic Structure of the Large Ribosomal Subunit at 2.4 Å Resolution" by Nenad Ban, Poul Nissen, Jeffrey Hansen, Peter B. Moore, Thomas A. Steitz Science 289, 905-920 (2000). http://bass.bio.uci.edu/~hudel/bs99a/lecture22/lecture3_5.html (1 of 2)5/25/2007 9:34:55 AM Supplemental Material (3.5) "The Structural Basis of Ribosome Activity in Peptide Bond Synthesis" by Poul Nissen, Jeffrey Hansen, Nenad Ban, Peter B. Moore, Thomas A. Steitz. Science 289, 920-930 (2000). These issues are available online or as hardcopies at the UCI Science Library. Next: Summary Please report typos, errors etc. by EMAIL (mention the title of this page). http://bass.bio.uci.edu/~hudel/bs99a/lecture22/lecture3_5.html (2 of 2)5/25/2007 9:34:55 AM Summary (3.6) Summary ● ● ● ● ● ● ● ● Ribosomes are large and complex molecular precision machines. Ribosomes occur free in the cytosol or attached to the endoplasmic reticulum. They contain two subunits, called small and large. Both subunits are comprised of large rRNAs and many proteins. rRNAs form the core and are stabilized by extended base-stacking & base-pairing. Most ribosomal proteins are located on the surface. Peptide-bond formation is catalyzed one base of the 23S rRNA (ribozyme) in the large subunit. Ribosomes have tunnels, channels and cavities which accommodate the various players during translation: mRNA, aminoacyl-tRNA, nascent protein. Next Lecture: Initiation of Protein Synthesis Please report typos, errors etc. by EMAIL (mention the title of this page). http://bass.bio.uci.edu/~hudel/bs99a/lecture22/lecture3_6.html5/25/2007 9:34:55 AM