Medicinal Biochemistry I Translation & Trafficking PDF 2024

Genetic Code Protein biosynthesis Protein trafficking Medicinal Biochemistry I Translation & Trafficking Žarko Bošković [email protected]...

Genetic Code Protein biosynthesis Protein trafficking Medicinal Biochemistry I Translation & Trafficking Žarko Bošković [email protected] KU MDCM October 9 – 21, 2024 1 / 60 Genetic Code Protein biosynthesis Protein trafficking Directionality of central dogma Figure: Francis Crick’s note on biological information transfer. 2 / 60 Genetic Code Protein biosynthesis Protein trafficking Genetic code Figure: Center nucleotide groups amino acids by property (to an extent). 3 / 60 Genetic Code Protein biosynthesis Protein trafficking Combinations of nucleotides encode specific amino acids There are 4 nucleotides. Q1: How many ways to arrange 4 different objects in 3 slots are there (if repetition is allowed)? Q2: What if repetition was not allowed? A1: 4x4x4 = 43 = 64 A2: 4x3x2 = 24 But there are only 20 amino acids. There’s redundancy in the code. 4 / 60 Genetic Code Protein biosynthesis Protein trafficking Where do we start and when do we finish? There are some special codons in the sequence to help with that. Initiation codon AUG Termination codon UAG (”amber”), UAA (”ochre”), UGA (”opal”) Every polypeptide begins with M. Remember, we are in the RNA world, so no T. List of codons, anticodons. 5 / 60 Genetic Code Protein biosynthesis Protein trafficking Complementarity of nucleotide bases Figure: Bases are matched and they can “recognize” its pair. 6 / 60 Genetic Code Protein biosynthesis Protein trafficking Base pairing and hydrogen bonds Figure: AT base pairing 7 / 60 Genetic Code Protein biosynthesis Protein trafficking Base pairing and hydrogen bonds Figure: GC base pairing 8 / 60 Genetic Code Protein biosynthesis Protein trafficking Genetic code expansion Hachimoji DNA: Figure 9 / 60 Genetic Code Protein biosynthesis Protein trafficking Translation Definition Translation is a process of synthesizing proteins by “translating” the nucleotide genetic code into amino acid sequence. 10 / 60 Genetic Code Protein biosynthesis Protein trafficking High level overview of translation Translation decodes the genetic information contained in mRNA. The major players in this are the mRNA, the tRNA (which has a 3 nucleotide anticodon loop and an amino acid acceptor end) and the ribosome. The prokaryotic ribosome consists of two subunits called the large and the small subunit. 11 / 60 Genetic Code Protein biosynthesis Protein trafficking Pattern in biopolymer syntheses Activation of precursors −−→ Initiation −−→ Elongation −−→ Termination −−→ Post – synthetic modifications 12 / 60 Genetic Code Protein biosynthesis Protein trafficking High level overview of translation 1. Activation of the precursors. 2. Initiation. The two components of the ribosome have to come together at the translation start site to form a functioning ribosome. 3. Elongation. tRNA must enter the ribosome and the anticodon must hybridize to the codon in the mRNA. Now the new amino acid can be ligated onto the nascent protein. Then the tRNA must move out of the way to make room for the next one. 4. Termination/recycling. When the ribosome reaches a stop codon, this will be recognized by a termination factor, allowing hydrolysis of the polypeptide chain. 13 / 60 Genetic Code Protein biosynthesis Protein trafficking Information transfer Figure: Central dogma of biology localized to organelles. 14 / 60 Genetic Code Protein biosynthesis Protein trafficking 15 / 60 Genetic Code Protein biosynthesis Protein trafficking Initiation Actors: mRNA (messenger RNA) N-formylmethionyl-tRNAfMet initiation codon on mRNA (AUG) 30S ribosomal subunit 50S ribosomal subunit GTP (i.e. not ATP) initiation factors (IF-1, IF-2, IF-3) Setting: ribosome 16 / 60 Genetic Code Protein biosynthesis Protein trafficking Elongation Actors: Fully formed ribosome (70S) aminoacyl-tRNA specified by codons elongation factors (EF-Tu, EF-TS, EF-G), GTP, Mg2+. Setting: Ribosome 17 / 60 Genetic Code Protein biosynthesis Protein trafficking Elongation Figure: Three arrangements of tRNA during elongation. YouTube video of this process. 18 / 60 Genetic Code Protein biosynthesis Protein trafficking Elongation 19 / 60 Genetic Code Protein biosynthesis Protein trafficking Termination and ribosome recycling Actors: Termination codon in mRNA release factors (RF-1, RF-2, RF-3), EF-G, IF-3 20 / 60 Genetic Code Protein biosynthesis Protein trafficking Activation of precursors Actors: amino acids amino acyl tRNA synthetase tRNA (transfer RNA) ATP Mg2+ Setting: cytosol (i.e. not ribosome) 21 / 60 Genetic Code Protein biosynthesis Protein trafficking Activation: “Charging” the amino acid onto its cognate tRNA 22 / 60 Genetic Code Protein biosynthesis Protein trafficking Activation: “Charging” the amino acid onto its cognate tRNA Reaction: AA + tRNAAA + ATP −−→ AA – tRNAAA + AMP + PP ∆G = −29kJmol −1 23 / 60 Genetic Code Protein biosynthesis Protein trafficking Important properties of tRNA 1. Specific binding to its amino acid 2. Recognizes codon on mRNA 3. T-loop interacts with the ribosome 4. D-loop interacts with tRNA syntetase 24 / 60 Genetic Code Protein biosynthesis Protein trafficking Key player: transfer RNA (tRNA) Definition Transfer ribonucleic acid (tRNA) is a type of RNA molecule that helps decode a messenger RNA (mRNA) sequence into a protein. Figure: Transfer RNA molecule. 25 / 60 Genetic Code Protein biosynthesis Protein trafficking Figure: mRNA-tRNA-peptide. 26 / 60 Genetic Code Protein biosynthesis Protein trafficking PyMOL exercise Aspartyl tRNA. PDB: fetch 1asy Phenylalanyl tRNA. PDB: fetch 4tna 27 / 60 Genetic Code Protein biosynthesis Protein trafficking Unusual bases in tRNA structure Special tRNA bases Figure: 5-Methylcytidine, 5,6-Dihydrouridine Figure: Pseudouridine vs. uridine. 28 / 60 Genetic Code Protein biosynthesis Protein trafficking Ribosomes synthesize proteins Figure: Transmission Electron Micrograph of ribosomes on mRNA. Groups of ribosomes attached to the same mRNA are called polysomes. 29 / 60 Genetic Code Protein biosynthesis Protein trafficking Ribosomes synthesize proteins, but many other species are involved Some facts about translation: 70 proteins and 20 enzymes, extra auxiliary enzymes, and other factors. 90% of cellular energy is used for this process. There are around 15,000 ribosomes in a cell. Synthesis of an average 100 amino acid protein takes about 5 seconds. Synthesis is coordinated with targeting and destruction. 30 / 60 Genetic Code Protein biosynthesis Protein trafficking Ribosomes 31 / 60 Genetic Code Protein biosynthesis Protein trafficking Ribosomal proteins Some facts about ribosomal proteins: Proteins found in the small subunit are prefixed S; those in the large subunit are prefixed L. Ribosomal proteins have various roles: stabilizing rRNA accelerating peptide bond formation (L27 and L16) and participating in extra-ribosomal functions no proteins are proximate to the amino acid transferase center of the ribosome 32 / 60 Genetic Code Protein biosynthesis Protein trafficking Ribosome Definition Ribosome is an assembly of proteins and nucleic acids that catalyzes peptide bond formation in nascent proteins. Source: BioNinja Figure: Ribosome at various levels of resolution 33 / 60 Genetic Code Protein biosynthesis Protein trafficking Ribosome Ribosomes are composed partly of RNA (rRNA) The small subunit contains the 16S rRNA subunit, which is 1542 nt The large subunit contains the 23S (2900 nt) and 5S (120 nt) rRNAs All of these rRNAs have well-defined secondary structures 34 / 60 Genetic Code Protein biosynthesis Protein trafficking Ribosome Functions of the large and the small subunit are now understood as follows: Small (30S): binds initiation factors binds initiator tRNA tRNA decoding/fidelity Large (50S): binds elongation factors binds the aminoacyl-tRNA extremity peptide bond formation 35 / 60 Genetic Code Protein biosynthesis Protein trafficking Polysomes Figure: Synchronous translation. 36 / 60 Genetic Code Protein biosynthesis Protein trafficking Shine-Dalgarno sequence Pyrimidine-rich sequence on ribosomes that establishes contact with purine-rich sequence on mRNA. 37 / 60 Genetic Code Protein biosynthesis Protein trafficking PyMOL exercise ribosome in complex with mRNA, and 3 tRNAs: fetch 4v4j 30 S subunit: fetch 4b3r Ribosome 30S subunit How many metal ions per subunit are there? What types of metal ions? How many different proteins? How many different nucleic acids? Can you identify the codon on mRNA fragment? Can you identify the anticodon on tRNA stem loop? What is the structure of the small molecule bound to it? 38 / 60 Genetic Code Protein biosynthesis Protein trafficking Messenger RNA Definition An mRNA molecule carries a portion of the DNA code to other parts of the cell for processing. mRNA is created during transcription. During the transcription process, a single strand of DNA is decoded by RNA polymerase, and mRNA is synthesized. Physically, mRNA is a strand of nucleotides known as ribonucleic acid, and is single-stranded. 39 / 60 Genetic Code Protein biosynthesis Protein trafficking Messenger RNA Figure: Schematic structure of a fully processed mRNA. Source: Daylite, Wikimedia 40 / 60 Genetic Code Protein biosynthesis Protein trafficking Which is larger, mRNA or the protein it codes for? Figure: mRNA is much larger. Source: David Goodsell. Nucleotides are more massive than amino acids (300 Da vs. 110 Da), there are 3 nucleotides encoding 1 amino acids, and there are regions of mRNA called introns that do not encode amino acids. 41 / 60 Genetic Code Protein biosynthesis Protein trafficking Wobble hypothesis Q: Is there one tRNA for each amino acid codon? A: No. Concept: wobbly base. Third base in a codon is “optional” Anticodons go from 3′ to 5′. 42 / 60 Genetic Code Protein biosynthesis Protein trafficking Antibiotic translation inhibitors Most antibiotics are inhibitors of prokaryotic ribosomes, in fact, almost all of them target the elongation cycle. For most drugs, at least one of the resistance mechanisms is mutations in the targeted RNA/protein. Puromycin Tetracyclines Chloramphenicol Streptomycin Cycloheximide 43 / 60 Genetic Code Protein biosynthesis Protein trafficking Antibiotic translation inhibitors Drug: Puromycin Mechanism of action Acting as an analog of the 3’ terminal end of aminoacyl-tRNA, puromycin incorporates itself into a growing polypeptide chain and causes its premature termination, thereby inhibiting protein synthesis. 44 / 60 Genetic Code Protein biosynthesis Protein trafficking Antibiotic translation inhibitors Drug: Tetracyclines Mechanism of action Binds to 30S subunit and inhibits binding of aminoacyl-tRNAs in prokaryotes. PyMOL: fetch 1hnw 45 / 60 Genetic Code Protein biosynthesis Protein trafficking Antibiotic translation inhibitors Drug: Chloramphenicol Mechanism of action Inhibits the peptidyl transferase activity of the 50S ribosomal subunit in prokaryotes. PyMOL: fetch 1nji 46 / 60 Genetic Code Protein biosynthesis Protein trafficking Antibiotic translation inhibitors Drug: Streptomycin Mechanism of action Inhibits initiation and causes misreading of mRNA in prokaryotes. PyMOL: fetch 4b3r 47 / 60 Genetic Code Protein biosynthesis Protein trafficking Antibiotic translation inhibitors Drug: Cycloheximide Mechanism of action Inhibits peptidyl transferase activity of the 60S subunit in eukaryotes. 48 / 60 Genetic Code Protein biosynthesis Protein trafficking Cell organization Figure: Animal cell. 49 / 60 Genetic Code Protein biosynthesis Protein trafficking Co-translational and post-translational transport Figure: Diagram of trafficking in cells. Source: Khan academy, based on Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., and Walter, P. (2002). A simplified “roadmap” of protein traffic. In Molecular biology of the cell (4th ed.). New York, NY: Garland Science. 50 / 60 Genetic Code Protein biosynthesis Protein trafficking Secretory and membrane proteins Made by ribosomes bound to ER. Figure: Endomembrane system of a eukaryotic cell. Source: Mariana Ruiz Villarreal, WikiMedia 51 / 60 Genetic Code Protein biosynthesis Protein trafficking Localization sequence on N-terminus is a zip code Figure: Sequences directing proteins for secretion or incorporation into membrane. 52 / 60 Genetic Code Protein biosynthesis Protein trafficking What happens in the ER doesn’t stay in the ER Figure: Rough ER is “studded” with ribosomes. Source: BruceBlaus, WikiMedia 53 / 60 Genetic Code Protein biosynthesis Protein trafficking ER-Golgi-Membrane Golgi apparatus exists in 3 forms: cis Golgi (near to ER) middle Golgi trans Golgi (far from ER). It can be in a form of cisternae Figure: Transport to membrane. or vesicles. 54 / 60 Genetic Code Protein biosynthesis Protein trafficking Ribosomes need to be first brought to the ER Figure: Signal recognition particle ensures ribosome gets to the ER. 55 / 60 Genetic Code Protein biosynthesis Protein trafficking Ribosomes need to be first brought to the ER Figure: Signal recognition particle ensures ribosome gets to the ER. 56 / 60 Genetic Code Protein biosynthesis Protein trafficking Journey of the transmembrane proteins 57 / 60 Genetic Code Protein biosynthesis Protein trafficking Protein degradation Figure: Proteasome: a protein recycle bin 58 / 60 Genetic Code Protein biosynthesis Protein trafficking Protein degradation Figure: Components of a proteasome. 59 / 60 Genetic Code Protein biosynthesis Protein trafficking Inhibition of protein degradation Drug: Bortezomib Mechanism of action Bortezomib reversibly inhibits the 26S proteasome, a large protease complex that degrades ubiquinated proteins. By blocking the targeted proteolysis normally performed by the proteasome, bortezomib disrupts various cell signaling pathways, leading to cell cycle arrest, apoptosis, and inhibition of angiogenesis. PyMOL: fetch 5lf3 60 / 60