5/8/06 Translation animation of translation http://www.blc.arizona.edu/INTERACTIVE/DNA3/protsyn95.mov How does the translation process work? How does this chemical conversion work? After the discovery of mRNA and the elucidation of the genetic code, molecular biologists puzzled over how a particular triplet of nucleotides could chemically code for a particular amino acid: In other words, how does a UGG codon ensure that the amino acid tryptophan is inserted into a polypeptide at a particular position in the polymer? 1 Some scientists suggested that an amino acid R group interacted directly with its corresponding codon (triplet) via a “pocket” formed on the template that was complementary in shape and charge (or could form complementary H-bonds) to the amino acid side group 2 BUT this idea was difficult to reconcile with the chemisty of nucleic acids and some of the R groups of amino acids Recall the various type of R groups found in amino acids: For which amino acids does this model make sense? For which amino acids does this model not make much sense? 3 4 Francis Crick and the adaptor hypothesis Crick proposed a solution to this problem by envisioning an adapter molecule attached to each amino acid that would interact directly via hydrogen bonding with a triplet codon animation of translation showing adaptor http://www.blc.arizona.edu/INTERACTIVE/DNA3/protsyn95.mov In essence Crick proposed the existence of a chemical go- between that was attached to an amino acid during protein synthesis and interacted via Hydrogen bonding with the codon on the mRNA 5 Francis Cricks hypothetical adaptor molecule (bridging the chemical gap between RNA and protein) turned out to be the tRNA (transfer RNA) molecule tRNA is the chemical go-between that allows amino acids to “interact” with an mRNA template tRNA's as adaptors: Each amino acid has at least one tRNA Each tRNA reads the codon or codons specific for one amino acid For example tRNAser has an anticodon that reads only serine codons Before it participates in protein synthesis, a tRNA is “charged” with its corresponding amino acid 6 REMEMBER that the CODE table is always presented in mRNA language CODON: mRNA language ANTI-CODON tRNA language LIKE OTHER H-BONDING INTERACTIONS BETWEEN NUCLEIC ACID POLYMERS, THE mRNA AND tRNA H- BOND IN AN ANTIPARALLEL FASHION 7 Transfer RNAs are the chemical interpreters in translation • A “charged” tRNA is a nucleic acid on one end: capable of forming hydrogen bonds with an mRNA molecule • It is an amino acid on the other end: capable of being incorporated into a polypeptide A charged tRNA acts as a chemical interpreter because it speaks in both chemical languages. This tRNA is charged with the amino acid cysteine. Via its AGC anti-codon, it can base-pair with the cysteine codon UGC in an mRNA 8 What does a tRNA look like? Figure 17.14 in text • Transfer RNAs are short RNA polymers ranging from 75- 85 nucleotides in length • Despite their small size, tRNAs have a fairly elaborate secondary and tertiary structure In contrast mRNAs have little secondary structure 9 Secondary structure • involves complementary base-pairing between different portions of the molecule to form stem-loop structures held together by short stretches of intra-strand h-bonds • All tRNAs have the same overall 2o and 3o structure but they differ in the sequence of the anticodon 10 Tertiary structure • similar to proteins, the tertiary structure is produced by folding the tRNA to produce a 3-D molecule with a characteristic L shape • at one end of the L is a loop of polymer which contains 3 bases called the anticodon • the anticodon hydrogen bonds with the corresponding codon on the mRNA • at the other end of the molecule is an attachment site for the amino acid Like proteins the tertiary structure of tRNAs is critical for their role in protein synthesis In contrast to proteins, all tRNAs fold up into the same shape NOTE: Genes that specify tRNA’s do not have a protein product: their final product is an RNA molecule 11 12 DNA TCA 5' 3' AGT transcription TCA 5' 3' 5' 3' 3' UCA 5' AGT splicing and processing serine codon in eukaryotes on mRNA mRNA UCA AGU serine anticodon 3' 5' on tRNA 5' serine serine attached to tRNA ser at 3' end Chemical conversion of TCA into serine. Accuracy of translation depends on precise matching: (1) of an amino acid with its cognate tRNA (2) of the anitcodon of a charged tRNA with its corresponding codon on the mRNA 13 Peptide bond formation always takes place on the surface of complex structures called ribosomes The ribosome orchestrates peptide bond formation: 1. It binds the charged tRNA and mRNA in the proper spatial orientation 2. This binding facilitates accurate decoding of the mRNA and the rapid formation of the peptide bond 3. It contains the polypeptide polymerase (real name is peptidyl transferase) which catalyzes peptide bond formation 14 The two essential functions of the ribosome: • The small subunit contains the messenger RNA decoding site • the large subunit the peptidyl transferase centre. • The mRNA threads through the subunit interface, and is decoded. • The three transfer RNA binding sites, A (acceptor), P (peptidyl) and E (exit) then handle amino-acid selection, addition and completion of polypeptide synthesis. Here a charged tRNA is shown in the A site, and a nascent peptidyl-tRNA in the P site; the E site is vacant. 15 Essential cell biology: animation of translation 07.6 The ribosome catalyzes formation of the peptide bond The catalytic function responsible for peptide bond formation is called peptidyl transferase The incorporation of an amino acid into a growing polypeptide chain. the polypeptide grows by stepwise addition of amino acids to its C-terminus. The figure shows the addition of the 4th amino acid to the chain. 16 The ribosome • is a particle that is found in abundance in all cells that are actively making protein • most complex component of the protein sythesizing machinery of the cell • made of ribosomal RNA (called rRNA) and a large number of proteins • rRNA has elaborate tertiary structure formed by intrastrand hydrogen bonds 17 Ribosomes in the cytoplasm of a eukaryotic cell. This electron micrograph shows a thin section of cytoplasm. The ribosomes appear as black dots (red arrows). Some are free in the cytosol. Others are attached to the endoplasmic reticulum 18 • Rough ER has ribosomes attached to the membrane surface • these ribosomes produce proteins that will be secreted by the cell (such as insulin in the pancreas) Essential cell biology: animation of polyribosomes 07.7 19 A comparison of the structure of eukaryotic and prokarytoic ribosomes. Despite the differences in size (indicated by the S value) both prokaryotic and eukaryotic ribosomes have nearly the same structure and they function similarly 20 See Figure 17.18 in text: Elongation of Polypeptides during translation NOTE: A, P and E sites NOTE; energy source is GTP, a relative of ATP 21 Science 285: 2077 9/24/99 Rotund marvels. Structure of the 70S ribosome and its functional center. (Top) The tRNA molecules span the space between the two subunits; the channel in the 50S subunit through which the growing peptide chain protrudes is shown in dashed lines. (Bottom) The 30S (left) and 50S (right) subunits have been opened up to give a better view of the three binding sites for tRNA, the A, P, and E sites. The 30S subunit shows the approximate location of the site where the codons of the mRNA are read by the anticodons of the tRNAs. The 50S subunit has the tRNA sites shown from the opposite direction. The acceptor ends of the A- and P-site tRNAs are close to each other in the peptidyl transfer site, which is close to the exit channel located behind a ridge in the 50S subunit. The binding site for EF-G and EF-Tu is located on the right-hand protuberance of the 50S subunit. 50S = large subunit 30s = small subunit (for prokaryotes) 22 1/28/02 Ribosomes and ribozymes prokaryotic ribosome -- 3 bound tRNAs rRNAs: cyan (16S), gray (23S) and light purple (5S) A, P, E site tRNAs in yellow, orange, red (respectively) small subunit proteins: dark purple large subunit proteins: magenta rRNA - ribosomal RNA tRNA = transfer RNA\ 23 All ribosomes contain a large and small subunit. Each subunit is made of various polypeptides and rRNA (ribosomal RNA) 24 For many years, the rRNA component of the ribosome was viewed as an inert structural matrix for the proteins that carried out the catalytic business of the ribosome Conventional wisdom said all biological catalysts are made of protein So biochemists spent many years looking for the catalytic activity among the various ribosomal proteins But they ran into a problem: they couldn’t seem to assign this function to a particular protein Discovery that RNAs could act as catalysts in an unrealted process set a precedent for thinking about rRNA as the business end of the ribosome -- the catalytic componant The discovery of RNA catalysis changed the way many scientists viewed the role of RNA in the ribosome Perhaps the proteins in the ribosome provide an inert structural matrix for catalytic RNA ribozyme: an RNA molecule that functions as a catalyst 25 MAKING THE PEPTIDE BOND Figure 2 Interactions of the CCA ends of ribosome-bound tRNA with the large ribosomal subunit. a, Cut-away view of the large ribosomal subunit with tRNAs bound. tRNAs are positioned on the large ribosomal subunit as described in the legend for Fig. 1, and the subunit sliced in half along a plane approximately perpendicular to the Fig.