Case 3 - Lost in Translation - PDF

Case 3 - Lost in translation 1. What are the codons and how do they work - Codon: A codon is a DNA or RNA sequence of three nucleotides (a trinucleotide) that forms a unit of genomic information encoding a particular amino acid or signaling the termination of protein synthesis (stop...

Case 3 - Lost in translation 1. What are the codons and how do they work - Codon: A codon is a DNA or RNA sequence of three nucleotides (a trinucleotide) that forms a unit of genomic information encoding a particular amino acid or signaling the termination of protein synthesis (stop signals). - 64 different codons → 61 specify amino acids + 3 are stop signals Characteristics of amino acids - Side chain determines identity - Dependent on side chain amino acids are: - non-charged: Apolar (hydrophobic), polar - charged: acid (polar, negatively charged), basic (polar, positively charged) - Zwitter ions → both a negative and positive charge - All amino acids are dissolvable in watery solutions - Iso-electric point (pl): pl is the pH which a molecule in a solution has no charge 2. Structure of ribosome and different types - Two main body parts Prokaryotes: - Small one: 30S subunit (S is a Svedberg unit used to indicate the weight of molecules) - Large one: 50S subunit - Subunits made up of rRNA + protein molecules → complement each other in achieving function of protein synthesis - 50S → two RNA molecules (23S and 5S ribosomal RNA), ribosomal proteins encasing it - 30S → carefully folded RNA molecule (16S ribosomal RNA), between the 30S proteins - The individual small and large subunit parts are synthesized inside the nucleus of a cell that, once they’re in the cytoplasm, assemble into one 70S ribosome unit - In the core of ribosome → 3 sites → main action 1. First, there is the driver A site (aminoacyl-tRNA site), that allows the entry of new tRNA molecules attached to an amino acid. 2. Next to it is the middle passenger P site (peptidyl-tRNA site), that holds a growing protein chain and the intermediate tRNA molecule in place. 3. And finally, the front passenger E site (the exit site) allows the exit of both the used tRNA molecules and the newly made protein chain out of the ribosome. Essentially, the small subunit part of the machine is for recognizing and correctly binding the incoming tRNA molecules and the large part is for facilitating the protein synthesis action. Differences prokaryotes and eukaryotes In bacteria, they are either free floating in the cytoplasm or attached to the plasma membrane. In eukaryotes they are either free floating in the cytoplasm or attached to the membrane of the endoplasmic reticulum (ER). E. coli contains about 16,000 ribosomes, whereas most eukaryotic cells have more than one million. Bacteria assemble their ribosomal subunits in the cytoplasm, and eukaryotes in the nucleolus. Poly ribosomes - Multiple copies from 1 mRNA by simultaneous translation 3. Translation itself the process Eukaryotes vs. Prokaryotes - Prokaryotes → polycistronic: 1 gene codes for multiple proteins direct translation in cytoplasm - Eukaryotes → monocistronic: 1 gene codes for one protein transport of mRNA out of the nucleus before translation Transfer RNA - The molecule is folded into a cloverleaf structure, with three base-paired stem portions and three loops with unpaired bases. - Connect mRNA codons to amino acids they encode - One end has an anticodon → can bind to specific mRNA codons - Other end carries the amino acid - The 3′ terminus is the attachment site for an amino acid. It ends with the sequence CCA, and the amino acid is bound to the ribose of the last nucleotide. - About 80 nucleotides long - At least 61 tRNAs would be needed for the 61 amino acid coding codons - However, most bacteria have fewer than 61 different tRNAs, and human mitochondria have only 22. A cytoplasmic aminoacyl-tRNA synthetase attaches an amino acid to the 3′ end of the tRNA, thereby converting it into an aminoacyl-tRNA. The ester bond between amino acid and tRNA is almost as energy rich as a phosphoanhydride bond in ATP, but the reaction is nevertheless irreversible because the pyrophosphate is quickly hydrolyzed in the cell. This is possible because the rules of base pairing are relaxed for the third codon base. Uracil at the 5′ end of the anticodon can pair not only with adenine (A) but also with guanine (G) at the 3′ end of the codon, and a G in this position can pair with cytosine (C) or uracil (U). This freedom of base pairing is called wobble. Steps of translation To see how cells make proteins, let's divide translation into three stages: initiation (starting off), elongation (adding on to the protein chain), and termination (finishing up). Initiation prokaryotes - To position the mRNA correctly on ribosome → Shine-Dalgarno sequence in the 5′-untranslated region of the mRNA (consensus: AGGAGGU), about 10 nucleotides upstream of the AUG start codon. - It base pairs with a complementary sequence on the 16S RNA in the small ribosomal subunit - This positions AUG on P site → where the anticodon of the initiator tRNA can base pair with the AUG codon while binding to the small ribosomal subunit - The initiator tRNA carries the modified amino acid N -formylmethionine (fMet). Therefore all bacterial proteins are synthesized with fMet at the amino terminus. This 30S initiation complex also contains three initiation factors. - IF-3 prevents binding of the large ribosomal subunit - The GTP-bound IF-2 is required for binding of fMet-tRNA to the 30S initiation complex (During the elongation stage of translation, GTP is used as an energy source for the binding of a new amino bound tRNA to the A-site of the ribosome. It is also used as an energy source for the translocation of the ribosome towards the 3' end of the mRNA.) - IF-1 prevents the binding of an additional tRNA to the complex. The 70S initiation complex is formed when the 50S ribosomal subunit binds to the 30S initiation complex. The initiation factors are released while the GTP that is bound to IF-2 is hydrolyzed to GDP and inorganic phosphate. The initiator tRNA binds to the P site , which is occupied by a p eptidyl-tRNA during the elongation phase. The A site (A = (a)minoacyl-tRNA, or (a)cceptor) is still empty. It receives incoming aminoacyl-tRNAs during the elongation phase. Initiation eukaryotes The Kozak consensus sequence (Kozak consensus or Kozak sequence) is a nucleic acid motif that functions as the protein translation initiation site in most eukaryotic mRNA transcripts. Regarded as the optimum sequence for initiating translation in eukaryotes, the sequence is an integral aspect of protein regulation and overall cellular health as well as having implications in human disease. It ensures that a protein is correctly translated from the genetic message, mediating ribosome assembly and translation initiation. A wrong start site can result in non-functional proteins. The initiation of protein synthesis appears to be devoted to the assembly of the catalytic rRNA and an initiator tRNA at the correct AUG codon of a template mRNA. These RNAs may now be widely acknowledged as the stars but they nevertheless cannot perform alone. Indeed the translation machinery, especially in eukaryotes, has evolved to require a host of protein factors. The focus of this review is on the essential parts played by the eukaryotic initiation factors (eIFs) in bringing together the initiator tRNA, ribosome and mRNA. eIF4E --> binding to cap In eukaryotes, at least eleven different initiation factors are required to properly initiate translation. Collectively, they ensure that the methionyl-initiator tRNA (Met-tRNAiMet) is brought in the P site of the ribosome to the initiator AUG of an mRNA. Conceptually, this process can be divided in four steps: 1. Formation of the 43S pre-initiation complex, when the Met-tRNAiMet is delivered by eIF2 to the P site of the 40S ribosomal subunit; 2. Recruitment of the 43S complex to the 5' end of the mRNA by eIF3 and the eIF4 factors; 3. Scanning of the 5' untranslated region (UTR) and recognition of the AUG codon 4. Assembly of the 80S ribosome Leaky scanning - Sometimes the first AUG is skipped → leaky scanning - Creates isoforms of the same protein - Proteins with/without signal peptide - various quantities of different isoforms Elongation - An aminoacyl-tRNA is placed into the A site of the ribosome by the GTP-binding elongation factor Tu (EF-Tu) - If (and only if) codon and anticodon match, the bound GTP is hydrolyzed to GDP, and EF-Tu with its bound GDP vacates the ribosome. - The aminoacyl-tRNA remains in the A site, ready for peptide bond formation. - The first peptide bond is formed when fMet is transferred from the initiator tRNA to the amino acid residue on the aminoacyl-tRNA in the A site. This reaction requires no external energy source because the free energy content of the ester bond in the fMet-tRNA (≈ 29 kJ/mol or 7 kcal/mol) exceeds that of the peptide bond (≈ 4 kJ/mol or 1 kcal/mol). - The peptidyl transferase of the large ribosomal subunit that catalyzes peptide bond formation is not a ribosomal protein, but it is an enzymatic activity of the 23S RNA in the large ribosomal subunit. Therefore the ribosome is an RNA enzyme, or ribozyme. Peptide bond formation leaves a free tRNA in the P site and a peptidyl-tRNA in the A site. The free tRNA moves from the P site to an E site (E = exit) on the large ribosomal subunit before leaving the ribosome altogether, and the peptidyl-tRNA moves from the A site into the P site. Codon-anticodon pairing remains intact. Therefore the ribosome moves along the mRNA by three bases. This step, called translocation, requires the GTP-binding elongation factor EF-G. Hydrolysis of the EF-G bound GTP is the energy source for translocation. The speed of ribosomal protein synthesis is about 20 amino acids per second, and the error rate is about 1 for every 10,000 amino acids. Termination - The stop codons UAA, UAG, and UGA have no matching tRNAs. Instead they are recognized by proteins called termination factors or release factors, which induce cleavage of the bond between polypeptide and tRNA. GTP hydrolysis takes place during translational termination. Eukaryotes Eukaryotic releasing factors are essential components in the process of translation termination in protein synthesis. They recognize stop codons in the mRNA and facilitate the release of the newly synthesized polypeptide chain from the ribosome. In eukaryotes, there are mainly two releasing factors involved: 1. eRF1 (Eukaryotic Release Factor 1): This factor recognizes all three stop codons (UAA, UAG, UGA) in mRNA. It plays a crucial role in the termination of translation by binding to the ribosome when a stop codon is encountered.  2. eRF3 (Eukaryotic Release Factor 3): This factor functions as a GTPase and works in conjunction with eRF1. It helps to stimulate the release of the polypeptide chain by facilitating the hydrolysis of GTP, which is necessary for the dissociation of the ribosomal subunits and the release of the newly synthesized protein. Together, eRF1 and eRF3 ensure that translation is properly terminated and that the polypeptide chain is released so that it can fold into its functional three-dimensional structure and perform its cellular functions. Prokaryotes In prokaryotes, the process of translation termination involves the following release factors: 1. RF1 (Release Factor 1): RF1 recognizes and binds to the stop codons UAA and UAG. It facilitates the release of the polypeptide chain from the ribosome when these stop codons are encountered. 2. RF2 (Release Factor 2): RF2 recognizes and binds to the stop codons UAA and UGA. Similar to RF1, it aids in the release of the polypeptide chain from the ribosome when these codons are present. 3. RF3 (Release Factor 3): RF3 is a GTPase that assists RF1 and RF2 in their function. It helps in the dissociation of the release factors from the ribosome and is involved in recycling them for subsequent rounds of translation termination. Together, RF1 and RF2 ensure that translation termination occurs at the correct stop codons, while RF3 supports the process by facilitating the release and recycling of the release factors. The Ribosome Recycling Factor (RRF) (prokaryotes) Function 1. Disassembly of the Ribosome: After the release factors (RF1 and RF2) have mediated the release of the newly synthesized polypeptide chain, the ribosome, which is now in a post-termination state, needs to be disassembled so its components can be reused for new rounds of translation. RRF assists in this process by helping to separate the large and small ribosomal subunits and remove the mRNA. 2. Recycling: Once the ribosome is disassembled, RRF, along with other factors like EF-G (Elongation Factor G) and sometimes IF3 (Initiation Factor 3), aids in recycling the ribosomal subunits and other components back into the pool of ribosomes available for new translation events. Mechanism - RF3-GDP kicks out releasing factors from A site → RF3 moves away as well  - RRF binds to the ribosome in the post-termination complex, which includes the mRNA, tRNA, and the ribosomal subunits. - RRF works in conjunction with EF-G, which is involved in the translocation of the ribosome during translation elongation. EF-G, in its GTP-bound form, assists in the conformational changes required for the release of the ribosomal subunits. - The combined action of RRF and EF-G leads to the release of the ribosomal subunits from the mRNA and each other, effectively disassembling the ribosome. 4. Regulation of translation Translation regulation is a crucial aspect of gene expression control in both prokaryotic and eukaryotic cells. It ensures that proteins are synthesized at the right time, in the right amounts, and under appropriate conditions. Here’s a summary of the primary mechanisms through which translation can be regulated: 1. Regulation at the mRNA Level - mRNA Stability: The stability of mRNA molecules affects their translation. Degradation of mRNA can be controlled by various factors, including RNA-binding proteins and microRNAs (miRNAs), which can lead to mRNA decay or stabilization. - 5' Cap and 3' Poly(A) Tail: In eukaryotes, the 5' cap structure and 3' poly(A) tail of mRNA are critical for translation initiation and stability. The length of the poly(A) tail can be regulated, impacting translation efficiency. - RNA Binding Proteins: Proteins that bind to specific sequences or structures in the mRNA can influence its translation. For example, binding proteins may block or facilitate ribosome binding. - Alternative Splicing: The production of different mRNA isoforms through alternative splicing can lead to variations in protein synthesis from the same gene. 2. Regulation at the Translation Initiation Stage - Initiation Factor Availability: Eukaryotic translation initiation involves several factors (eIFs). The availability and activity of these factors, such as eIF2 and eIF4E, can control the initiation of translation. - Phosphorylation: The phosphorylation of initiation factors (e.g., eIF2α) can inhibit or promote translation initiation. For example, stress conditions can lead to the phosphorylation of eIF2α, reducing overall protein synthesis but allowing for the selective translation of certain mRNAs. - Cap-Binding Proteins: In eukaryotes, proteins that bind to the 5' cap structure of mRNA can affect translation initiation. For instance, changes in cap-binding proteins can alter the efficiency of ribosome recruitment. 3. Regulation at the Translation Elongation and Termination Stages - Elongation Factors: The availability and activity of elongation factors (e.g., EF-Tu in prokaryotes and eEF1A in eukaryotes) influence the rate and accuracy of translation elongation. - Codon Usage: The presence of rare codons in mRNA can slow down translation elongation, impacting protein synthesis rates. Cells can regulate translation by modulating tRNA availability corresponding to these codons. - Regulation by Small RNAs: Small RNAs such as miRNAs and small interfering RNAs (siRNAs) can bind to complementary sequences in mRNA, leading to translation repression or degradation. 4. Regulation at the Post-Translational Level - Protein Modifications: After translation, proteins can be modified by processes such as phosphorylation, acetylation, or ubiquitination. These modifications can affect the protein’s stability, localization, and function, indirectly influencing translation regulation. In summary, translation can be regulated at multiple levels, including mRNA stability and modification, initiation factor activity, elongation efficiency, and response to cellular conditions. These regulatory mechanisms ensure that proteins are synthesized correctly and efficiently in response to the cell's needs and environmental changes. Lecture: 5. Protein folding maturation When protein folding into the mature, functional 3D state is complete, it is not necessarily the end of the protein maturation pathway. A folded protein can still undergo further processing through post-translational modifications. There are over 200 known types of post-translational modification, these modifications can alter protein activity, the ability of the protein to interact with other proteins and where the protein is found within the cell e.g. in the cell nucleus or cytoplasm. Through post-translational modifications, the diversity of proteins encoded by the genome is expanded by 2 to 3 orders of magnitude. There are four key classes of post-translational modification: 1. Cleavage - The resulting shortened protein has an altered polypeptide chain with different amino acids at the start and end of the chain. This post-translational modification often alters the proteins function, the protein can be inactivated or activated by the cleavage and can display new biological activities. 2. Addition of chemical groups - Following translation, small chemical groups can be added onto amino acids within the mature protein structure. Examples of processes which add chemical groups to the target protein include methylation, acetylation and phosphorylation. 3. Addition of complex molecules - Post-translational modifications can incorporate more complex, large molecules into the folded protein structure. One common example of this is glycosylation, the addition of a polysaccharide molecule, which is widely considered to be most common post-translational modification. 4. Formation of intramolecular bonds - Many proteins produced within the cell are secreted outside the cell to function as extracellular proteins. Extracellular proteins are exposed to a wide variety of conditions. To stabilize the 3D protein structure, covalent bonds are formed either within the protein or between the different polypeptide chains in the quaternary structure. The most prevalent type is a disulfide bond (also known as a disulfide bridge). Chaperone In molecular biology, molecular chaperones are proteins that assist the conformational folding or unfolding of large proteins or macromolecular protein complexes. There are a number of classes of molecular chaperones, all of which function to assist large proteins in proper protein folding during or after synthesis, and after partial denaturation. Chaperones are also involved in the translocation of proteins for proteolysis. 6. Disposal of proteins Protein digestion begins when you first start chewing. There are two enzymes in your saliva called amylase and lipase. They mostly break down carbohydrates and fats. Once a protein source reaches your stomach, hydrochloric acid and enzymes called proteases break it down into smaller chains of amino acids. Amino acids are joined together by peptides, which are broken by proteases. From your stomach, these smaller chains of amino acids move into your small intestine. As this happens, your pancreas releases enzymes and a bicarbonate buffer that reduces the acidity of digested food. This reduction allows more enzymes to work on further breaking down amino acid chains into individual amino acids. The Ubiquitin-Proteasome Pathway The major pathway of selective protein degradation in eukaryotic cells uses ubiquitin as a marker that targets cytosolic and nuclear proteins for rapid proteolysis (Figure 7.39). Ubiquitin is a 76-amino-acid polypeptide that is highly conserved in all eukaryotes (yeasts, animals, and plants). Proteins are marked for degradation by the attachment of ubiquitin to the amino group of the side chain of a lysine residue. Additional ubiquitins are then added to form a multiubiquitin chain. Such polyubiquinated proteins are recognized and degraded by a large, multisubunit protease complex, called the proteasome. Ubiquitin is released in the process, so it can be reused in another cycle. It is noteworthy that both the attachment of ubiquitin and the degradation of marked proteins require energy in the form of ATP. Proteins are marked for rapid degradation by the covalent attachment of several molecules of ubiquitin. Ubiquitin is first activated by the enzyme E1. Activated ubiquitin is then transferred to one of several different ubiquitin-conjugating enzymes (E2). In most cases, the ubiquitin is then transferred to a ubiquitin ligase (E3) and then to a specific target protein. Multiple ubiquitins are then added, and the polyubiquinated proteins are degraded by a protease complex (the proteasome). Maintaining appropriate levels of proteins within cells largely relies on a cellular component called the proteasome, which degrades unneeded or defective proteins to recycle the components for the eventual assembly of new proteins. Lysosomes The other major pathway of protein degradation in eukaryotic cells involves the uptake of proteins by lysosomes. Lysosomes are membrane-enclosed organelles that contain an array of digestive enzymes, including several proteases. They have several roles in cell metabolism, including the digestion of extracellular proteins taken up by endocytosis as well as the gradual turnover of cytoplasmic organelles and cytosolic proteins.