Fundamentals in Biology 1: From Molecules to the Biochemistry of Cells PDF

Script to accompany: Fundamentals in Biology 1: From Molecules to the Biochemistry of Cells This script is for internal ETH use only, not to be copied, distributed or sold. It contains referenced images from external sources and copyrighted new images....

Script to accompany: Fundamentals in Biology 1: From Molecules to the Biochemistry of Cells This script is for internal ETH use only, not to be copied, distributed or sold. It contains referenced images from external sources and copyrighted new images. Part 3: Translation TRANSLATION Introduction Today we will start our discussion on translation - the process of moving from one language of nucleic acids (cytosine, guanine, adenine and thymine) to the language of proteins (consisting of 20 different amino acids). We will discuss the genetic code, enzymes, and molecules that couple this information, as well as where in the cell translation takes place, and the machinery (ribosomes) that carries it out. The genetic code Translation is much more complex than DNA replication or transcription, since the alphabet of the language is changing from a four-letter alphabet (nucleotides) to a twenty-letter alphabet (amino acids). When calculating the number of nucleotide combinations from nucleic acid sequences of different lengths, it becomes apparent that to uniquely specify the 20 different amino acids, a sequence of at least 3 nucleotides is necessary (42=16, 43=64). This, however, implies that the genetic code is degenerate - meaning that there is more information than needed, as there are 64 possible sequences of three nucleotides made out of four letters. In other words, many of these codons would be expected to have the same meaning and specify the same amino acid. To understand how this language of triplets is read, geneticists performed experiments using simple systems (e.g. bacteriophage T4, a virus that infects bacteria). They used certain chemicals to introduce mutations in the DNA sequence that resulted in an addition or deletion of a base. They realized that by introducing an extra base and shifting the reading frame of triplet sequences makes the produced protein nonfunctional, or one can say it makes the message illegible. Using similar mutagenesis experiments, they observed that a single nucleotide deletion on a sequence that already had a single nucleotide insertion could restore the function of the protein, and this is an example of a suppressor mutation. THE BAN LAB AND ETH ARE FUN THE XBA NLA BAN DET HAR EFU N (frameshift mutation due to insertion) THE_ANL ABA NDE THA REF UN (frameshift mutation due to deletion) THE XBX ANX LAB AND ETH ARE FUN (local change, majority still in-frame) THE_ANL AXB AND ETH ARE FUN (suppressor mutation, local change) Effect of mutations on a protein. Frameshift mutations render the mRNA illegible, while other mutations lead to specific amino acid alterations, insertions or deletions, leaving the majority of the reading frame intact. A suppressor mutation brings a frameshift mutation back into frame. Ultimately it was shown by Marshal Nirenberg, Gobind Khorana, Robert Holley and others that the genetic code is read in groups of three, and is non-overlapping; meaning that once the reading frame is established, it does not change. Furthermore, the code is not random, meaning that a single nucleotide change often results in a different amino acid with similar chemical characteristics. Three codons (UGA, UAA and UAG) specify a stop, and one codon (AUG) specifies the start, encoding a methonine. By interpreting the meaning of all 64 different combinations of triplets, scientists were able to arrive at the genetic code table, which is the standard for most archaea, bacteria and the cytosolic translation of eukaryotes (in the meantime, specific alterations to the standard table have been mainly found in some unicellular organisms such as ciliates and in mitochondrial organelles). The standard table of the genetic code (here to be read from the central 5’to the peripheral 3’) reveals how the 64 possible base triplets encode for the 20 amino acids. The AUG codon, read as a methionine, can serve as START codon depending on the mRNA context, while the codons UGA, UAA and UAG are STOP codons, representing the signal for release of the growing peptide chain from the ribosome. (Modified after Wikipedia and www.labxchange.org) tRNA Adapter Molecules In order to understand how nucleotide triplets are interpreted to specify the sequence of amino acids in proteins, scientists hypothesized that there must be an adapter RNA molecule. This molecule would have to have a particular amino acid attached to it and should be capable of recognizing a triplet of codons according to the base pairing principle. It turned out that this molecule is a transfer RNA (tRNA), a type of RNA molecule that helps decode a messenger RNA (mRNA) sequence into a protein. It is made up of a specific sequence of nucleotides, typically between 75 and 90 nucleotides long. tRNAs are single stranded RNAs with complementary regions that can be visualized in a secondary structure diagram as a cloverleaf. Upon folding, tRNAs adopts an L-shaped three-dimensional structure that allows it to interact at the same time with the ribosome and the mRNA. It forms two arms, an acceptor arm where the amino acid is attached, and an anticodon arm containing an anticodon triplet, which is complementary to the codon in the mRNA. Cloverleaf and 3D representations of a tRNA. Note the directionality of the mRNA compared to the tRNA and the L-shape of the tRNA when folded, with two major arms. (Adapted from Brock, Biology of Microorganisms, 15th ed. 2019) Aminoacyl tRNA Synthetases Aminoacyl tRNA synthetase enzymes add a specific amino acid to the corresponding tRNA molecule. There are 20 different synthetases, one for each amino acid. These enzymes catalyze the formation of an ester bond between the AMP-activated amino acid and the 3’-hydroxyl group at the end of the tRNA molecule. To avoid mischarging of the tRNA with the wrong amino acid, each synthetase is able to specifically recognize the unique features of its tRNA (including the anticodon), as well as the corresponding amino acid. One of the 20 different aminoacyl tRNA synthetases (valyl-tRNA synthetase) charging tRNAVal specifically with its cognate amino acid using an AMP-activated valyl precursor to form Val-tRNAVal. (From Brock, Biology of Microorganisms, 15th ed. 2019) The Ribosome The ribosome is a complex molecular machine found in all living cells that functions as the site of protein synthesis. The ribosome binds to messenger RNA (mRNA), transfer RNA (tRNA), and other RNA molecules during the process of protein synthesis. The mRNA carries the instructions to make a particular protein, and the tRNA adapter molecules carry the amino acids that are used to build the protein. The ribosome reads the instructions from the mRNA using the tRNAs for decoding and to assemble the amino acids into a functioning protein. The bacterial ribosome is composed of two unequally sized, asymmetric subunits termed the 30S small and the 50S large subunit, respectively. Together they form the 70S ribosome. The sizes of the small and the large ribosomal subunits are expressed in Svedberg coefficients (S) according to their sedimentation behavior during centrifugation. They do not add up to the size of the associated ribosome (70S), since the Svedberg coefficient does not only depend on the molecular weight of the molecule but also on its shape. The 30S and 50S subunits are composed of both ribonucleic acids (ribosomal RNAs, rRNAs) and dozens of proteins. Under the negative stain electron microscope, the ribosome has a crown-like shape. The molecular weight of the 70S bacterial ribosome is 2.5 megadaltons (MDa). Enzymes are much smaller, usually around 25,000 daltons (25kDa). The small subunit of the ribosome is 930 kDa and the large subunit is 1.6 MDa. The small subunit comprises a 16S ribosomal RNA that is 1500 nucleotides long. The large subunit harbors a 23S rRNA that is nearly 3000 nucleotides and a 5S ribosomal RNA with 120 nucleotides. While the small subunit has 21 proteins, the large subunit contains 31. The relative ratio of protein to RNA mass in the ribosome is 2/3 or 66%, with only 34% of the mass contributed by proteins. This shows that ribosomes are primarily RNA-based machines. Note that the ribosome is actually an ancient ribozyme, with the small subunit rRNA scaffold harboring the regions of rRNA involved in decoding and the large subunit segments containing the rRNA residues of the catalytic site. Historical and recent structures of the bacterial ribosome. The architecture of the ribosome was derived from low resolution negative stain EM micrographs (here crosslinked using antibodies against the nascent protein chain). Recently, using latest detector technology and cryo-EM on state-of-the-art microscopes, the structure was determined to very high resolution. Stages of Protein Synthesis Protein synthesis on the ribosome occurs in three stages: initiation, elongation, and termination. Initiation begins with the small ribosomal subunit facilitating formation of contacts between the formylmethionine initiator tRNA (fMet-tRNAifMet) and the AUG start codon on a mRNA. Elongation follows with the successive addition of other amino acids (each carried by a tRNA molecule) to the growing chain, as guided by the mRNA sequence. Finally, termination occurs when the ribosome encounters one of the stop codons, which signals the end of the protein chain and leads to its release from the ribosome. Translation direction on the mRNA is from the 5' end to the 3' end, and proteins are synthesized with the N-terminal amino acid first. Initiation Stage of Translation Translation initiation is the process by which the small ribosomal subunit binds to a messenger RNA (mRNA) molecule to appropriately position the initiator tRNA. This process is catalyzed by several translation initiation factors (IFs). During translation initiation, the mRNA molecule is scanned for a specific sequence [termed Shine- Dalgarno sequence (SD) or ribosomal binding site (RBS)] that helps the 30S subunit position itself on the start codon, which is usually AUG. This codon is recognized by the initiator tRNA, which carries the amino acid formylmethionine and is delivered to the 30S subunit with the help of IF2 that hydrolyzes GTP in the process. Initiation factors 1 and 3 also help in the formation of the 30S initiation complex. At the end of initiation, the large ribosomal subunit joins the complex, the initiation factors dissociate, and the elongation stage can start. Bacterial translation initiation depends on IF1, IF2 and IF3, and one GTP is consumed for the delivery of the initiator tRNA onto the small subunit. Note which DNA-encoded regulatory elements are used during transcription by the DNA polymerase versus translation by the ribosome. (Modified after Brock, Biology of Microorganisms, 15th ed. 2019) Translation Elongation During the elongation stage of protein synthesis, the ribosome binds to the mRNA and moves along the mRNA strand, reading successive codons, and the ribosome catalyzes the joining of amino acids together based on the codon sequence. The process involves a transfer of the polypeptide chain from the peptidyl tRNA to the aminoacyl tRNA, such that the new amino acid is added to the C-terminal side of the growing polypeptide chain. The catalytic reaction is catalyzed by the RNA molecule that is part of the large ribosomal subunit and is called the peptidyl transferase reaction. The ribosome is therefore a ribozyme that catalyzes the transfer of a peptidyl moiety from a tRNA molecule to an amino acid. The peptidyl transferase reaction involves the formation of a covalent peptide bond between the carboxy carbon of the peptidyl-tRNA molecule in the P (peptidyl) site and the alpha-amino group of the amino acid on the acyl-tRNA in the A (aminoacyl) site. This process is repeated until the entire chain is formed. Elongation stage of protein synthesis. After the initiator tRNA is correctly positioned into the P-site and the reading frame is established, EF-Tu delivers the next (elongator) aminoacyl-tRNA into the A site. In the peptidyl transferase center (PTC) of the large ribosomal subunit, the amino group of the amino acid attached to the A site tRNA attacks the ester bond of the P-site residue, thereby reconnecting the growing peptide chain. EF-G then promotes the translocation of the ribosome along the mRNA by one codon, and the next tRNA can be positioned into the A site by EF-Tu. The elongation cycles continue in frame until a STOP codon reaches the A site. For each reaction cycle, two GTPs are hydrolyzed. (Modified after Brock, Biology of Microorganisms, 15th ed. 2019) Detailed view of the peptidyl transferase reaction. (Adapted from Stryer, Biochemistry, 8th Edition, Freeman 2015) Elongation factors EF-G, EF-Tu, and EF-Ts are protein factors that help the ribosome move along the mRNA strand and facilitate the elongation stage of translation. EF-G helps the ribosome move along the mRNA strand by hydrolyzing GTP. EF-Tu binds to incoming aminoacyl tRNA molecules and delivers them to the ribosome helping in codon recognition. Since EF-Tu also hydrolyzes GTP when delivering the tRNA to the ribosome, it needs help by a GDP to GTP exchange factor EF-Ts, the third translation elongation factor, to restore its active GTP conformation and participate in the next cycle of tRNA delivery. Termination of Translation Termination of protein synthesis occurs when the ribosome reaches a stop codon (UAA, UAG, or UGA) in the mRNA sequence. Release factors participate in this process by binding to the ribosome and recognizing the stop codon. Release factors then catalyze the hydrolysis of the peptidyl-tRNA bond, which leads to the release of the newly formed protein. The ribosomal subunits then dissociate from each other and the mRNA is released. Termination of translation by release-factor mediated STOP codon recognition and peptide cleavage to release the newly synthesized protein. After this, the components of the termination complex are recycled. (Modified after Brock, Biology of Microorganisms, 15th ed. 2019) Polysomes and Transcription-Translation Coupling During protein synthesis polysomes can form when two or more ribosomes attach to the same mRNA molecule and begin translating. They are made up of multiple ribosomes bound together by messenger RNA, and are responsible for the efficient production of proteins in cells. Schematic and experimental visualization of bacterial polysomes, formed by 70S ribosomes which are arranged along a mRNA that is being synthesized by RNA polymerase. (from Brock, Biology of Microorganisms, 15th ed. 2019, Xue et al., Nature 2022) Transcription precedes translation and it occurs in the 5' to 3' direction, meaning that the 5' end of the mRNA is synthesized first. Because translation also proceeds in the same 5’ to 3’ direction, and in bacteria occurs in the same cellular compartment, it can be coupled with transcription. Active coupling of transcription and translation, with the help of dedicated transcription factors, in bacteria is a process that increases the efficiency of protein production. Coupling of transcription and translation in bacteria, where both processes take place simultaneously in the same cellular compartment. (Modified after Kohler et al., Science 2017) Protein Targeting to Cellular Membranes Protein targeting is a crucial process in all living cells. In bacteria, many proteins are synthesized in the cytoplasm but need to be transported to different locations, such as the plasma membrane or the extracellular environment. This process is highly coordinated and involves several mechanisms to ensure accuracy and efficiency. SecA Protein and its Role One of the key components in bacterial protein targeting is the SecA protein that drives the post-translational translocation of proteins. Proteins destined for secretion typically have a 15-20 aa long hydrophobic signal sequence at their N-terminus, which is recognized by the Sec machinery. SecA binds to these precursor proteins and, using energy from ATP hydrolysis, helps to "push" them through the protein-conducting channel in the membrane. This process is critical for proper localization of many bacterial proteins, including enzymes, toxins, and cell surface components. Signal Recognition Particle (SRP) Another important player in protein targeting is the Signal Recognition Particle (SRP). In bacteria, the SRP is a ribonucleoprotein complex that recognizes and targets specific proteins to the plasma membrane during their synthesis. The SRP binds to signal sequences of nascent polypeptides emerging from the ribosome. This binding directs the translating ribosome to the SRP receptor on the membrane. Once there, the protein is handed over to the protein conducting pore and inserted into the membrane. This co-translational targeting is particularly important for integral membrane proteins and proteins that need to be properly folded in the membrane environment. The fate of newly synthesized proteins depends on the presence of a signal sequence (red). Proteins lacking a signal sequence remain in the cytosol, while those carrying an N-terminal targeting signal are either exported via SecA into the periplasm or inserted into the membrane. Note that membrane targeting depends on the signal recognition particle, which forms a complex with the ribosome and recognizes the signal sequence as soon as it emerges from the ribosomal exit tunnel (the sketch in the book is wrong). (Modified after Brock, Biology of Microorganisms, 15th ed. 2019)