At 3 am, on Saturday May 27, 1961, J Heinrich Matthaei began one of the most important experiments in the history of science They discovered that polyuridylate (poly U) could direct the synthesis of polyphenylalanine in vitro. By showing that UUU encodes phenylalanine, they identified the first codon.
► In principle, a genetic code made up of three-letter words can be either overlapping or non-overlapping. One of the non-overlapping advantages of a non-overlapping code is that each letter is part of only one word; therefore, mutating a single nucleotide affects only one codon All living organisms use a nonoverlapping genetic code The enigma cryptography machine used by German armed forces during the Second World War
The standard genetic code has several prominent features: features 1. The genetic code is unambiguous 2. genetic code is said to be degenerate. There are multiple codons for most amino acids. degenerate UCU and CGU both specify Ser; ACA, ACC, ACG, and ACU all specify Thr - synonymous codons 3. The first two nucleotides of a codon are often enough to specify a given amino acid. For example, the four codons for glycine (GGU, GGC, GGA, and GGG) all begin with GG 4. Codons with similar sequences specify chemically similar amino acids 5. Only 61 of the 64 codons specify amino acids. The three remaining codons (UAA, UGA, UAA UGA and UAG) are termination UAG codons, or stop codons. The codons methionine codon, AUG, specifies the initiation site for protein synthesis and is often called the initiation codon
Transfer RNA molecules are named for the amino acid they carry (t. RNAPhe) Conserved bases (gray)
► Much of the base pairing between the codon and the anticodon is governed by the rules of Watson-Crick base pairing: A pairs with U, G pairs with C, and the strands in the base-paired region are antiparallel ► However, some exceptions to these rules led Francis Crick to propose that complementary Watson-Crick base pairing is required for only two of the three base pairs formed - “wobble” mechanism and 5`-position of the anticodon wobble position wobble Inosinate (I: t. RNAAla) is often found at the 5` (wobble) position of a t. RNA anticodon IGC can bind to three different codons specifying alanine: GCU, GCC, and GCA Different t. RNA molecules that can attach to the same amino acid are called iso-acceptor t. RNA molecules Genome sequencing data reveal that bacterial genomes encode 30 to 60 different t. RNAs and that eukaryotic genomes have genes for as many as 80 different t. RNA molecules
► Like DNA and RNA synthesis, protein synthesis can be divided into three distinct stages: initiation, chain elongation, and termination + additional step - aminoacylation of t. RNA ► Each of the 20 amino acids is covalently attached to the 3` end of its respective t. RNA molecules. The product of this reaction is called an aminoacyl-t. RNA ► Most species have at least 20 different aminoacyl-t. RNA synthetases in each cell since there are 20 different amino acids. A few species have two different aminoacyl-t. RNA synthetases for the same amino acid ► Some bacteria don’t have glutaminyl- or asparaginyl t. RNA synthetases ► Although each synthetase is specific for a particular amino acid, it can recognize many iso-acceptor t. RNA molecules. For example, there are six codons for serine and several different t. RNASer iso-acceptor molecules. All these different t. RNASer molecules are recognized by the organism’s single seryl-t. RNA synthetase enzyme
► The amino acid is covalently attached to the t. RNA molecule by the formation of an ester linkage between the carboxylate group of the amino acid and a hydroxyl group of the ribose at the 3` end of the t. RNA molecule. Since all t. RNAs end in -CCA the attachment site is always an adenylate residue ► The Gibbs free energy of hydrolysis of an aminoacylt. RNA is approximately equivalent to that of a phosphoanhydride bond in ATP. The energy stored in the aminoacyl-t. RNA is ultimately used in the formation of a peptide bond during protein synthesis ► Attaching a specific amino acid to its corresponding t. RNA is a crucial step in translating a genetic message. If there are errors at this step, the wrong amino acid could be incorporated into a protein
► Each aminoacyl-t. RNA synthetase binds ATP and selects the proper amino acid based on its charge, size, and hydrophobicity. Tyrosyl-t. RNA synthetase almost always binds tyrosine but rarely phenylalanine or any other amino acid The error rate for most aminoacyl-t. RNA synthetases is low because they make multiple contacts with a specific t. RNA and a specific amino acid. However, isoleucine and valine are chemically similar amino acids, and both can be accommodated in the active site of isoleucyl-t. RNA synthetase Isoleucyl-t. RNA synthetase mistakenly catalyzes the formation of the valyl-adenylate intermediate about 1% of the time The observed substitution of valine for isoleucine in polypeptide chains is only about 1 time in 10, 000
► During polypeptide chain elongation the ribosome and associated components move, or translocate, translocate along the template m. RNA in the 5`→ 3` direction ► The polypeptide is synthesized from the N-terminus to its C-terminus ► All ribosomes contain two subunits of unequal size. In E. coli, the small subunit is called the 30 S (5. 5 ◦ 22. 5 nm) subunit and the large subunit is called the 50 S (15 ◦ 20 nm) subunit. 50 S subunit also contains a tunnel about 10 nm long and 2. 5 nm in diameter. The 30 S and 50 S subunits combine to form an active 70 S ribosome (prokaryote) prokaryote Both prokaryotic and eukaryotic genomes contain multiple copies of ribosomal RNA genes 160 n 2904 nucleotides 120 nucleotides 1542 nucleotides Eukaryotic ribosomes are similar in shape to bacterial ribosomes but they tend to be somewhat larger and more complex
Peptidyl site (P site), forming peptidyl-t. RNA site The second aminoacyl-t. RNA is bound at the aminoacyl site (A site) site ► The initiation of protein synthesis involves assembling a translation complex at the beginning of an m. RNA’s coding sequence. This complex consists of the two ribosomal subunits, an m. RNA template to be translated, an initiator t. RNA molecule, and several accessory proteins called initiation factors. This crucial initiation step ensures that the proper initiation codon factors (and therefore the correct reading frame) is selected before translation begins
► The first codon translated is usually AUG. Every cell contains at least two types of AUG methionyl-t. RNAMet molecules that can recognize AUG codons: initiator (t. RNAf. Met in bacteria, t. RNAi. Met in eukaryotes and archaebacteria) and internal t. RNA ► Although these two molecules have different primary sequences, and distinct functions, both of them are aminoacylated by the same methionyl-t. RNA synthetase ► The charged initiator t. RNA is the substrate for a formyltransferase that catalyzes addition of a formyl group from 10 -formyltetrahydrofolate to the methionine residue producing N-formylmethionyl-t. RNAf. Met (f. Met-t. RNAf. Met) ► The methionine that begins protein synthesis in eukaryotes is not formylated ► There are several possible reading frames in an m. RNA molecule but only one of them is correct. Establishing the correct reading frame during the initiation of translation is critical for the accurate decoding of information from m. RNA into protein. It is important to appreciate that the initiation codon is not simply the first three nucleotides of the 5`-end m. RNA. Initiation codons can be located many nucleotides downstream of the m. RNA molecule
► In prokaryotes, the selection of an initiation site depends on an interaction between the small subunit of the ribosome and the m. RNA template. The 30 S subunit binds to the m. RNA template at a purine-rich region just upstream of the correct initiation codon. This region, called the Shine-Dalgarno sequence, is complementary to a pyrimidine-rich stretch at the 3` end of the 16 S r. RNA molecule. During formation of the initiation complex, these complementary nucleotides pair to form a doublestranded RNA structure that binds the m. RNA to the ribosome. The result of this interaction is to position the initiation codon at the P site on the ribosome. The initiation complex assembles exclusively at initiation codons because Shine-Dalgarno sequences are not found immediately upstream of internal methionine codons Ribosome-binding sites at the end of m. RNA for several E. coli proteins. The Shine-Dalgarno sequences (red) occur immediately upstream of initiation codons (blue). Complementary base pairing between the 3` end of 16 S r. RNA and the region near the 5` end of an m. RNA. Binding of the 3` end of the 16 S r. RNA to the Shine-Dalgarno sequence helps establish the correct reading frame for translation by positioning the initiation codon at the ribosome’s P site.
► Formation of the initiation complex requires several initiation factors in addition to ribosomes, initiator t. RNA, and m. RNA ► Prokaryotes contain three initiation factors, designated IF-1, IF-2, and IF-3 IF-1 IF-2 ► There at least eight eukaryotic initiation factors (e. IF’s) e. IF’s ► In both prokaryotes and eukaryotes, the initiation factors catalyze assembly of the protein synthesis complex at the initiation codon IF-3 - maintains the ribosomal subunits in their dissociated state by binding to the small subunit. The ribosomal subunits bind separately to the initiation complex and the association of IF-3 with the 30 S subunit prevents the 30 S and 50 S subunits from forming the 70 S complex prematurely - IF-3 also helps position f. Met-t. RNAf. Met and the initiation codon at the P site of the ribosome IF-2 selects the initiator t. RNA from the pool of aminoacylated t. RNA molecules in the cell. It binds GTP forming an IF-2–GTP complex that specifically recognizes the initiator t. RNA and rejects all other aminoacyl-t. RNA molecules IF-1 binds to the 30 S subunit and facilitates the actions of IF-2 and IF-3 Once the 30 S complex has been formed at the initiation codon, the 50 S ribosomal subunit binds to the 30 S subunit
► Eukaryotic m. RNAs DO NOT have distinct Shine-Dalgarno sequences that serve as ribosome binding sites. The first AUG codon in the message usually serves as the initiation Codon e. IF-4 - cap binding protein (CBP), binds specifically to the 7 -methylguanylate cap at the 5` end of eukaryotic m. RNA. Binding of e. IF-4 to the cap structure leads to the formation of a pre-initiation complex consisting of the 40 S ribosomal subunit, an aminoacylated initiator t. RNA, and several other initiation factors. The preinitiation complex then scans along the m. RNA in the 5`→ 3` direction until it encounters an initiation codon ► When the search is successful, the small ribosomal subunit is positioned so that Mett. RNAi. Met interacts with the initiation codon in the P site. In the final step, the 60 S ribosomal subunit binds to complete the 80 S initiation complex and all the initiation factors dissociate. The dissociation of e. IF-2—the eukaryotic counterpart of bacterial IF-2— is accompanied by GTP hydrolysis ► m. RNA molecules that encode several polypeptides are said to be polycistronic (in bacteria)
► The initiator t. RNA occupies the P site in the ribosome and the A site is ready to receive an incoming aminoacyl-t. RNA three-step microcycle (1) Positioning the correct aminoacyl-t. RNA in the A site of the ribosome (2) forming the peptide bond (3) shifting, or translocating, the m. RNA by one codon relative to the ribosome (the two t. RNAs in the ribosome’s P and A sites also translocate) The translation machinery works relatively slowly compared to the enzyme systems that catalyze DNA replication (18 amino acid residues ps, replisomes synthesize DNA at a rate of 1 K nucleotides ps, transcription in prokaryotes is approximately 55 nucleotides ps) This difference in rates reflects, in part, the difference between polymerizing four types of nucleotides to make nucleic acids and polymerizing 20 types of amino acids to make proteins. Testing and rejecting all of the incorrect aminoacylt. RNA molecules also takes time and slows protein synthesis ► In bacteria, translation initiation occurs as soon as the end of an m. RNA is synthesized and translation and transcription are coupled (polyribosome or polysome). This tight polysome coupling is not possible in eukaryotes because transcription and translation are carried out in separate compartments of the cell
An E. coli cell contains about 20, 000 ribosomes. Many large eukaryotic cells have several hundred thousand ribosomes. A site is empty and the P site is occupied by the aminoacylated initiator t. RNA The first step in chain elongation is insertion of the correct aminoacyl-t. RNA into the A site of the ribosome. In bacteria, this step is catalyzed by an elongation factor called EF-Tu is a monomeric protein that contains a binding site for GTP. Each E. coli cell has about 135, 000 molecules of EF-Tu, making it one of the most abundant proteins in the cell EF-Tu–GTP associates with an aminoacyl-t. RNA molecule to form a ternary complex that fits into the A site of a ribosome. Almost all aminoacyl-t. RNA molecules in vivo are found in such ternary complexes
► The structure of EF-Tu is similar to that of IF-2 (which also binds GTP) and other G proteins , suggesting that they all evolved from a common ancestral protein ► The EF-Tu–GTP complex recognizes common features of the tertiary structure of t. RNA molecules and binds tightly to all aminoacyl-t. RNA molecules except f. Met-t. RNAf. Met. The molecule is distinguished from all other aminoacyl-t. RNA molecules by the distinctive secondary structure of its acceptor stem ► A ternary complex of EF-Tu–GTP–aminoacyl-t. RNA can diffuse freely into the A site in the ribosome. When correct base pairs form between the anticodon of the aminoacylt. RNA and the m. RNA codon in the A site, the complex is stabilized. ► EF-Tu–GTP can then contact sites in the ribosome as well as the t. RNA in the P site. These contacts trigger hydrolysis of GTP to GDP and causing a conformational change in EF-Tu– GDP that releases the bound aminoacyl-t. RNA. EF-Tu–GDP then dissociates from the chain elongation complex. ► EF-Tu–GDP cannot bind another aminoacyl-t. RNA molecule until GDP dissociates. An additional elongation factor called EF-Ts catalyzes the exchange of bound GDP for GTP
GDP/GTP substitution EF-Ts
Note that one GTP molecule is hydrolyzed for every aminoacylt. RNA that is successfully inserted into the A site
► Binding of a correct aminoacyl-t. RNA in the A site aligns the activated amino acid’s a-amino group next to the ester bond’s carbonyl on the peptidyl-t. RNA in the neighboring P site ► The nitrogen atom’s lone pair of electrons execute a nucleophilic attack on the carbonyl carbon, resulting in the formation of a peptide bond via a displacement reaction ► The peptide chain, now one amino acid longer, is transferred from the t. RNA in the P site to the t. RNA in the A site. Formation of the peptide bond requires hydrolysis of the energyrich peptidyl-t. RNA linkage ► The enzymatic activity responsible formation of the peptide bond is referred to as peptidyl transferase. This activity is contained within the large ribosomal subunit. Both the transferase 23 S r. RNA molecule and the 50 S ribosomal proteins contribute to the substrate binding sites, but the catalytic activity is localized to the RNA component. Thus, peptidyl transferase is yet another example of an RNA-catalyzed reaction.
translocation ► After the peptide bond has formed, the newly created peptidyl-t. RNA is partially in the A site and partially in the P site ► The deaminoacylated t. RNA has been displaced somewhat from the P site. It now occupies a position on the ribosome that is referred to as the exit site, or E site ► In prokaryotes, the translocation step requires a third elongation factor, EF-G. Like the other EF-G elongation factors, EF-G is an abundant protein; an E. coli cell contains approximately 20 K molecules of EF-G ► Binding of EF-G–GTP to the ribosome completes the translocation of the peptidylt. RNA from the A site to the P site and releases the deaminoacylated t. RNA from the E site. EF-G itself is released from the ribosome only when its bound GTP is hydrolyzed to GDP and Pi is released. The dissociation of EFG–GDP leaves the ribosome free to begin another microcycle of chain elongation HSP 70
The elongation reactions in eukaryotes are very similar to those in E. coli 3 chain elongation factors (CEFs): CEFs ● EF-1 a - docks the aminoacyl-t. RNA in the A site; its activity thus parallels that of E. coli EF-Tu ● EF-1 b - acts like bacterial EF-Ts, recycling EF-1 a ● EF-2 carries out translocation in eukaryotes (EF-G analogue) EF-Tu and EF-1 a are highly conserved, homologous proteins, as are EF-G and EF-2 ► Eukaryotic and prokaryotic ribosomal RNAs are also very similar in sequence and in secondary structure. These similarities indicate that the common ancestor of prokaryotes and eukaryotes carried out protein synthesis in a manner similar to that seen in modern organisms. Thus, protein synthesis is one of the most ancient and fundamental biochemical reactions. ► E. coli has three release factors (RF-1, RF-2, and RF-3). After formation of the final RF-1 RF-2 RF-3 peptide bond, the peptidyl-t. RNA is translocated from the A site to the P site, as usual. The translocation positions one of the three termination codons (UGA, UAG, or UAA) in the A UGA UAG UAA site. These termination codons are not recognized by any t. RNA molecules so protein synthesis stalls at the termination codon. ► Eventually, one of the release factors diffuses into the A site. RF-1 recognizes UAA and UAG and RF-2 recognizes UAA and UGA. RF-3 binds GTP and enhances the effects of RF-1 and RF-2. When the release factors recognize a termination codon, they cause hydrolysis of the peptidyl-t. RNA
streptomycin, chloramphenicol, erythromycin, and tetracycline, are specific for bacteria and have little or no effect on eukaryotic protein synthesis macrocyclic lactone Streptomycin Launched - 1943 Merck & Co. Chloramphenicol Launched Pfizer interacts with the 50 S subunit and inhibits peptidyl transferase 16 S and 30 S Ribosomal Protein Inhibitor inhibits the initiation of translation Erythromycin Launched – 1952 Abbott binds to the 50 S subunit, inhibiting the translocation step Minocycline Launched – 1971 Pfizer 30 S Ribosomal Protein Inhibitor Prevents the binding of aminoacyl-t. RNA molecules to the A site
► The N-terminal residues start to fold into the native protein structure even before the Cterminus of the protein has been synthesized. As these residues fold, they are acted on by enzymes that modify the nascent chain ► Modifications that occur before the polypeptide chain is complete are said to be cotranslational, whereas those that occur after the chain is complete are said to be posttranslational Co-translational and post-translational modifications include: ● de-formylation of the N-terminal residue ● removal of the N-terminal methionine ● formation of disulfide bonds ● cleavage by proteinases ● phosphorylation ● addition of carbohydrate residues (glycosylation, glycoproteins) ● acetylation ► One of the most important events that occurs co- and post-translationally is the processing and transport of proteins through membranes. In fact, proteins are synthesized by membrane bound ribosomes that are attached to the plasma membrane in bacteria and to the endoplasmic reticulum in eukaryotic cells
In cells that make large amounts of secreted protein, the endoplasmic reticulum membranes are covered with ribosomes
signal peptides ► Signal peptides vary in length and composition, but they are typically from 16 to 30 residues long and include 4 to 15 hydrophobic residues
In eukaryotes 1. 80 S initiation complex—including a ribosome, a Met -t. RNAi. Met molecule, and an m. RNA molecule—forms in the cytosol 2. ribosome begins translating the m. RNA and synthesizing the signal peptide at the N-terminus of the precursor 3. Once the signal peptide has been synthesized and extruded from the ribosome, it binds to a protein. RNA complex called a signal recognition particle (SRP) SRP - SRP is a small ribonucleoprotein containing a 300 nucleotide RNA molecule called 7 SL RNA and four proteins 4. The transport of proteins across the membrane is assisted by chaperones in the lumen of the endoplasmic reticulum. In addition to their role in protein folding, chaperones are required for translocation, and their activity requires ATP hydrolysis.
► Many integral membrane proteins and secretory proteins contain covalently bound oligosaccharide chains. The addition of these chains to proteins is called protein glycosylation. Protein glycosylation is one of the major metabolic activities of the lumen of the endoplasmic reticulum and of the Golgi apparatus and is an extension of the general process of protein biosynthesis ► The mass of the carbohydrate portion may account for as little as 1% or as much as 80% of the mass of the glycoprotein ► A common glycosylation reaction involves the covalent attachment of a complex oligosaccharide to the side chain clip of an asparagine residue ► In some cases, the structure of the oligosaccharide acts as a signal to target proteins to a specific location. For example, lysosomal proteins contain sites for the attachment of an oligosaccharide that targets these proteins to the lysosome
ВОПРОС № 4 Известно, что это ЛС (Mw=258. 24) является ингибитором продукции TNF-a, цитохрома P 450 CYP 3 A 4 и других белков. На сегодняшний день оно используется в некоторых странах для химиотерапии различных опухолевых заболеваний, несмотря на то, что в начале 60 -х годов оно было отозвано с рынка, где применялось с целью снижения воспалительных, рвотных реакций и бессонницы. Соединение содержит хиральный атом углерода, в организме человека обнаруживаются оба оптических изомера (R и S). Ниже приведен один из метаболитов этого соединения и H 1 спектр исходной молекулы. Известно, что это ЛС можно синтезировать из фталевого ангидрида (или 1, 2 -диметилового эфира 2 -карбокси бензойной кислоты) и L-глутамина в одну стадию (in situ). Финальный продукт легко алкилируется по атому азота различными алкилгалогенидами и вступает в реакцию с формальдегидом, образуя N-(метилгидрокси)-производное. Назовите это ЛС, приведите структурную формулу, объясните по какой причине оно было отозвано с рынка? MII