2022-23 WEEK 29 Protein synthesis.pdf

Module BIOL22081: Biomolecular Structure and Function WEEK 29. 15/ February /2023 Protein synthesis in the ribosomes: proofreading mechanism ALDO • Protein synthesis in the ribosomes • Protein folding and stability • Molecular basis of allosteric mechanisms • Allosteric control: case...

Module BIOL22081: Biomolecular Structure and Function WEEK 29. 15/ February /2023 Protein synthesis in the ribosomes: proofreading mechanism ALDO • Protein synthesis in the ribosomes • Protein folding and stability • Molecular basis of allosteric mechanisms • Allosteric control: case studies • Protein miss folding • Protein aggregation • Protein refolding in the cell Next series of lectures in this module: PROTEINS WEEKS 29 to 35 Protein synthesis in the ribosomes: learning outcomes At the end of this lecture you should be able to: • Explain the importance of hydrogen bonds in reading the genetic code • Discuss the structure and activation mechanism of transfer RNA and its importance as a translator molecule • Explain the role of aminoacyl tRNA synthetase in securing the fidelity of information transfer from DNA into proteins • Explain the proof -reading mechanism carried out by the elongation factor Tu (EF -Tu) and the role of GTP as energy -provider A brief reminder from BIOL14404 Introduction to Biochemistry Dr Craig Doig. DNA’s alphabet : .... just 4 letters A, C, G and T A molecule’s SHAPE define its PARTNERS Linear hydrogen bonds Linear hydrogen bonds A molecule’s SHAPE define its PARTNERS DNA RNA From DNA to RNA information transfer is relatively easy due to 1 - to - 1 complementary base pairs, so DNA acts as a TEMPLATE mRNA A BIG PROBLEM : Amino acids do not align to nucleic acids. So, mRNA cannot be a direct TEMPLATE Amino acids A Translator molecule is needed This translator molecule must be able to recognize BOTH nucleic acids and amino acids. Nucleic acids Amino acids Transfer RNA: a translator molecule 1 - transfer RNA: translating the genetic code protein synthesis in the ribosomes Duration: 8 minutes Loading of right amino acid by the enzyme Aminoacyl tRNA synthase “the fidelity of this step is extremely high, which is critical for the overall fidelity of protein synthesis ” Prokaryotes: Shine -Delgarno sequence anchors AUG codon in the P site Eucaryotes: initiating transport RNA is already in the initial binding complex Elongation factor (EF -TU) and proofreading mechanism “Thermodynamics and kinetics mechanisms are used to aid in discrimination such that the system chooses with extreme fidelity the cognate (i.e. right) tRNA from the 30 or 40 different tRNA available in the cell ” ATP -dependent translocation to free A binding site for a new incoming tRNA 27 April 2023 18 “ Hemoglobin synthesis in real time” This is not a live web link. The file with this animation is in NOW module page . The file can be downloaded and saved in your own pc. Animation Transfer RNA: a translator molecule This region recognizes 3 RNA’s nucleic acids (A, U, G and C bases) This region recognizes 1 out of 20 amino acids amino acid attached to tRNA Complementary codon ( 3 bases) An example : Tryptophan ACC tRNA tryptophanyl tRNA + tryptophan This reaction is catalysed by tryptophanyl tRNA synthase. The shape of this enzyme is complementary to both: ACCtRNA and tryptophan . How the genetic code works : an example 1 ..... CCG UAA UGG UCA GUG ..... mRNA U G G An mRNA segment contains the UGG codon for the amino acid tryptophan. 2 ACC -transfer RNA recognizes and binds tryptophan ONLY. 3 ACC tryptophanyl tRNA is formed This reaction is catalysed by tryptophanyl tRNA synthase. The shape of this enzyme is complementary to both: ACCtRNA and tryptophan . 4 ..... CCG UAA UGG UCA GUG ..... mRNA U G G C C A The ACC of tRNA aligns to UGG because they are complementary base pairs !!!! The ACC tryptphanyl tRNA aligns to UGG because they have complementary base pairs !!!! Self -study Aminoacyl tRNA synthetase mechanism: a summary Duration: 2 minutes 2 - Ribosomes’ proof reading mechanism 27 April 2023 30 “Ribosomes proof - reading mechanism” This is not a live web link. The file with this animation is in NOW module page. The file can be downloaded and saved in your own pc. Animation A proofreading mechanism: Elongation factor Tu (EF - Tu) EF - Tu A G G = Arginine A proofreading mechanism: Elongation factor Tu (EF - Tu) A G G C C U A proofreading mechanism: Elongation factor Tu (EF - Tu) Because CCA is not the right complementary codon , the interaction is weak (dissociation rate is fast ). Therefore there is no time for GTP to hydrolize and release the wrong amino acid . The wrong complementary codon CCA A proofreading mechanism: Elongation factor Tu (EF - Tu) Because UCC is the right complementary codon, the interaction is strong (dissociation rate is slow ). Therefore this allows time for GTP to hydrolize and release the right amino acid !!!! . The right complementary codon UCC A proofreading mechanism: Elongation factor Tu (EF - Tu) Using conformational work to pull the ribosome: Elongation factor G (EF - G) Free energy from GTP hydrolysis is used to produce conformational work resulting in the small subunit moving forward. GTP hydrolysis: GTP  AGP + Pi + BIOL14404 Introduction to Biochemistry: Equilibrium and rate constants & Enzyme Kinetics lectures x 1 ?????? �?????? = ?????? ?????? ?????? �� ?????? ��� = ?????? ?????? association + + di ssociation What would happens if k on for all aminoacyltRNA is SMALL? S ampling until finding the right amynoacyltRNA would take a long time. In the cell, k on depends on molecular size and shape. k on is bound to be roughly similar to all 20 aminoacids association + How is the value for k off expected to be for the right aminoacyl tRNA? Big or small? Why? The k off value for the right aminoacyltRNA is expected to be small . This is due to the strong interaction of the complementary hydrogen bonds between aminoacyltRNA and mRNA’s codon. Dissociation is not favoured . + di ssociation 3 - Protein synthesis rates: What can we learn from it? From DNA to RNA : transcription From DNA to DNA : DNA replication From RNA to aminoacids : translation 800 bases per second 50 bases per second 2 amino acids per second Why is protein synthesis so slow ??? 800 > 50 > 2 Where is the bottleneck ??? There are 20 different amino acids There are 64 codons in mRNA There are ~30 different aminoacyltRNAs . There are ~20 different aminoacyl tRNA synthases Doing the numbers: For example, for tryptophan, only 1 codon is the right one. So, ribosome have to sample many of them before getting the right one!!! Protein synthesis in the ribosomes: CONCLUSIONS • Complementarity of hydrogen bonds between messenger RNA and transfer RNA is fundamental for ensuring the correct reading of the genetic code. • transfer RNA act as a translator molecule by recognizing both: amino acids and nucleic acids (codons). • Aminoacyl tRNA synthetase is key on securing that the right amino acid is attached to the specific transfer RNA molecule • The proof -reading mechanism by the elongation factor Tu (EF -Tu) relies on the strength of the interaction of the hydrogen bonds formed between the right codons and anticodons. • Codon complementarity is reflected in low dissociation constant values ( K off )