BIO206 Lecture 15 Chapter 7.pdf

Lecture 15/ Chapter 7 November-04-13 8:44 PM Ribosomes Composition and structure, rRNA transcription Functional sites: A, P, E, EF-Tu, EF-G, mRNA Translation Principles Initiation Elongation Termination Quality Control There are 3 stages in initiation, the main differences between prokaryotes and eukaryotes happen in Stage 2 Translation The process by which the base sequence of mRNA is used to order and join the amino acids in a protein. Involves over 100 macromolecules 3 types of RNA Messenger RNA  mRNA Ribosomal RNA  rRNA  ribosomes Transfer RNA  tRNA  aminoacyl tRNA Aminoacyl-tRNA synthetases (aaRS) - transcription was called transcription because DNA to RNA is making a new copy using the same alphabet (except for uracil instead of thymidine). “transcribe” is writing a copy - translation implies converting to another language - in this case the 4 base-pair code of RNA has to be converted to the 20 amino acid code of proteins. - macromolecules are isolated or associated in complexes - one of these is RIBOSOMES which are complexes of proteins and RNA - three types of RNA also involved Order of amino acid is defined by nucleotide in mRNA rRNA - Catalyzes formation of peptide bond tRNA - Bring substrate to ribosome • Substrate = aa • Product = peptide Covalent bond - peptide bond between 2 amino acid EF-Tu - elongation factor thermo unstable - mRNA interacts with the ribosome in specific binding, -there are 3 sites where tRNA can bind. Stylized version is good for discussing function but we will look at the actual shape first Can see the A site, the P site and the E site on left are represented by specific colours on the right - A is pink, P is green, E is yellow Red chain of beads is the mRNA strand threaded through the sites A site – aa first interacts with ribosome P site – where peptide bond is formed P site – where peptide bond is formed E-site – final resting place where tRNA associates with ribosome before it exits mRNA binding site – location where mRNA interacts with ribosome and associates with codon recognition with A site and P site If EF-Tu binds, EF-G cannot – they are overlap each other – mutually exclusive sites To complete A and P sites, both subunits need to be present mRNA Binding to the 30S Complex How does a ribosome find an mRNA? Shine-Dalgarno sequence associates with 3’ end of 16S rRNA. • Binding allows ribosome to select proper initiation codon. Shine-Dalgarno sequences of mRNA • 3-10 nucleotides long • Purine-rich (A, G) consensus sequence is AGGAGG • Partly complementary to 3’ end of 16S rRNA • Anti-Shine-Dalgarno sequence is located at the 3' end of the 16S rRNA – CCUCCU • 16 nucleotides upstream of AUG start site • One for each translation start site (polycistronic) - the binding at the SD sequence permits the ribosome to pick the correct start site for translation - ribosomes with altered anti S-D sequences have greatly reduced mRNA recognizing ability. They can translate efficiently HOWEVER if the SD sequence in a gene has been mutated to match the altered ribosome. - also if you cleave the 3’ end of 16s rRNA you get a ribosome that can elongate and terminate but cant initiate new chain synthesis. -the SD sequences can be in between genes as here or even be inside the previous gene (ie not a spacer) since some bacterial genes overlap. How ribosome finds mRNA and recognizes it How prokaryotes translate mRNA Polycistronic mRNA Ribosome binding site – allows initiation of protein synthesis – found upstream (also called Shine-Dalgarno sequence) – a consensus sequence AGGAGG – can bind to Anti-Shine Dalgarno CCUCCU Polycistronic usually have single ribosome binding Monocistronic – multiple ribosome binding site Prokaryotic Initiation - Stage 1 Inactive ribosome dissociation • Soluble protein Initiation Factors • IF-1 and IF-3 • Extra-ribosomal proteins • Transient component of ribosome • NECESSARY for re-initiation of translation (ribosomes recycled) - ribosome subunits at the end of translation are left associated with each other. The complex is INACTIVE - to return to activity from inactivity the subunits must dissociate. - 2 initiation factors (1, 3) are involved in this, they stay associated with the 30s subunit after the large subunit has dissociated • this is a continuously occurring cycle, so that is why there is a “pool” of subunits in the cell as I said earlier. Reactivation process of inactive ribosome is needed to be translated again – this requires initiation factors Prokaryotic Initiation - Stage 2 Formation of 30S initiation complex • 30S subunit +IF-1 + IF-3 GTP • GTP • mRNA (Shine-Dalgarno) (AGGAGGN 8-15UG) • fMet-tRNA fMet • Binds at P-site • IF-2 SD = RBS = Complementary to 3' end of the 16S rRNA - the GTP, mRNA, and tRNA-formylated met IN COMPLEX with initiation factor 2 assemble with the small subunit/initiation factors and form the 30s initiation complex. GTP is the source of energy for protein synthesis. Using GTP, not ATP allows cell to segregate metabolism. The mRNA binds at the Shine-Delgarno sequence, interacting with the 16s rRNA. The first AUG ends up with the initiating tRNA associated The initiating tRNA is charged with methionine that, in prokaryotes, is FORMYLATED. Also the initiating tRNA is distinct from the regular met-tRNA. SAME AMINOACYL tRNA SYNTHETASE FOR THIS AND REGULAR tRNA. Polycystronic mRNA for prokaryotes - more than one shine-delgarno sequence (sometimes can be within the previous gene) First substrate is tRNA, identifies AUG- codon anti codon recognition The position AUG corresponds to where P site is – only time ribosome will entry P-site, all other entries in A site fMET-tRNA fMET - Methionine and charged with methionine What is fMet ? Formyl methionine – methyl with addition of formate group to the amino nitrogen of backbone Two tRNA: fMET, methionine aa that are incorporated to protein body Prokaryotic Initiator tRNA - ribosome subunits at the end of translation are left associated with each other. The complex is INACTIVE - to return to activity from inactivity the subunits must dissociate. Stage 1 - IF-1,IF-3 binding and dissociation of large subunit Stage 2 -the 30s complex is bound by initiating charged Met-tRNA, the mRNA (interacts with ribosome at Shine-dalgarno sequence), GTP and IF-2 THIS IS WHERE MOST OF THE DIFFERENCES WITH EUKARYOTIC TRANSLATION ARE Stage 3- in order the steps listed happen - IF-3 released, 50s subunit binds, hydrolysis of GTP gives conformational change and other Ifs released. End up with 70S complex with the initiating tRNA in the P SITE Aminoacylsynthetase – charges methionine Prokaryotic Initiation - Stage 3 Formation of 70S initiation complex • IF-3 release • 50S subunit joins • GTP hydrolyzed • Rearrangement of 30S complex • Rearrangement of 30S complex • IF-1 + IF-2 released The fMet-tRNA fMetoccupies the P site - the steps listed here happen in order - the GTP hydrolysis allows the conformational change that releases the initiation factors, and the 70s initiation complex is ready (Not straight energy bond like ATP) First amino acid in P-site = fMET Prokaryotic Initiation: Summary Stage 1: Inactive ribosome dissociation • IF-1, IF-3 • NECESSARY for translation re-initiation Stage 2: Formation of 30S initiation complex • 30S + IF-1 + IF-3 • fMET-tRNA fMet+ mRNA (S.D.) • GTP • IF-2 Stage 3: Formation of 70S initiation complex • IF-3 release  50S  GTP hydrolyzed  Rearrangement of 30S  IF-1 + IF-2 released fMet The fMet-tRNA occupies the P site - ribosome subunits at the end of translation are left associated with each other. The complex is INACTIVE - to return to activity from inactivity the subunits must dissociate. Stage 1 - IF-1,IF-3 binding and dissociation of large subunit Stage 2 -the 30s complex is bound by initating charged Met-tRNA, the mRNA (interacts with ribosome at Shine-dalgarno sequence), GTP and IF-2 THIS IS WHERE MOST OF THE DIFFERENCES WITH EUKARYOTIC TRANSLATION ARE Stage 3- in order the steps listed happen - IF-3 released, 50s subunit binds, hydrolysis of GTP gives conformational change and other Ifs released. End up with 70S complex with the initiating tRNA in the P SITE Prokaryotic Chain Elongation • Synthesis N  C terminus • Sequential amino acid addition to C-terminus • Peptide bond formed • Requires GTP • Requires non-ribosomal protein elongation factors (EF) EF – transiently binds to ribosome Translation: Elongation Initiation Elongation Stage 1 - Binding Stage 2 - Transpeptidation Stage 3 - Translocation Termination synthesis N  C terminus IT IS A POLYMER = Sequential amino acid addition up to 40 amino acids/second requires G(recycling of elongation factors) requires non-ribosomal elongation factors (EF) Chain Elongation - Binding 3 sequential steps Step 1: Binding aa ○ EF-Tu + aa-tRNA + GTP formed ○ Complex binds at A-site ○ GTP  GDP, EF-Tu released ○ aa-tRNA remains bound ONLY codon-anticodon interaction selects aa-tRNA aa - there is a repeated 3 step cycle for elongating the chain. Basically the same for prokaryotes and eukaryotes - this diagram is for FIRST elongation - Remember in protein structure we talked about EF-TU, bacterial elongation factor. This is where it acts • EF-Tu helps tRNA-aa complex associate with the ribosome. Complex forms, binds at the A site, GTP hydrolysis allows release of EF-Tu to go and find another tRNA NOT ENERGY, CONFORMATION - remember LOTS of copies of EF-Tu if the anticodon does not match then the amino acid does not stay bound. • could have errors if the wrong aa is attached to tRNA - no other method for proofreading here. • Codon anti codon binding – step 1 • How does A site select the right tRNA • aa-tRNA is not the substrate for the rxn – it is a larger complex (EF-Tu) associates with GTP that is the substrate – binds to small subunit ribosome • This binding is kinetic reaction – binds and releases until it finds the right one • Beginning of codon anticodon recognition • GTP is hydrolyzed and EF-Tu released – an irreversible biochemical reaction EF-Tu / aatRNA / GTP: Substrate • Part of bacterial ribosomal complex: binds tRNA • GTP bound  “latch” closed  tRNA bound • GTP  GDP  “latch” opens  tRNA released • GDP released (EF-Ts)  ready for new GTP and tRNA - there is a “latch” holding the domains of the protein together - when GTP is bound this latch is closed, and the tRNA binds to the ribosome with the help of the factor - the hydrolysis of GTP to GDP causes a small conformational change (only a few H atoms movement) but this is magnified by subsequent shifts throughout the protein - the “latch” is released and subunits can twist apart - tRNA is released and is free to be ejected later • GDP release with the help of other elongation factors EF-Ts and GTP binding ready EF-Tu for the next tRNA, and protein synthesis proceeds EF-Tu- a multidomain protein • It is segregated to 3 separate domain that