protein synthesis

editing

The aminoacyl-tRNA synthetase carries out this editing step to ensure against misacylated tRNA being used in protein synthesis. • Any tRNA bearing the wrong amino acid would be used for protein synthesis, possibly causing the synthesis of a harmful protein. • Editing of aminoacyl-tRNAs increases the overall accuracy of tRNA charging to approximatly 1 mistake in 40,000 couplings. • As bond formation uses energy which is then wasted as the bond is recleaved by the same enzyme this is known as a futile cycle. • However mistakes in this process are so dangerous to the cell that the expenditure is worth it.

Translation

The conversion of the RNA message to a protein needs information to be converted to a whole new language where different units are used. - Where as there are 4 bases in both DNA and RNA there are 20 different amino acids in proteins.

Termination of translation

Translation ends when one of three stop codons, UAA, UAG, or UGA, enters the A site of the ribosome. • There are no aminoacyl tRNA molecules that recognize these sequences. • Instead, stop codon's are recognized by release factors (RF's). • RF1 = UAA or UAG RF2 = UAA or UGA • These RF's trigger the hydrolysis of the ester bond in the peptdyltRNA causing the polypeptide to be transferred to a molecule of water releasing it from the tRNA. see diagram

Redundancy

• As we have seen the genetic code exhibits some redundancy. • 61 codons encode 20 amino acids. • Therefore there must either be more than 20 tRNA's or 1 tRNA will recognize multiple codons. • Both of these situations are true. - Bacteria have 31 different tRNA's - Humans have 48 different tRNA's

Initiation of Translation - Eukaryotes

• Unlike prokaryotes, eukaryotic mRNA undergoes posttranscriptional RNA modifications. • Whereas prokaryotic initiation begins with the recognition of the ribosome binding site on the mRNA, eukaryotic initiation begins with the ribosomal recognition of the 5' cap.

Mechanisms of Antibiotic Resistance

1. Enzymatic destruction of drug 2. Prevention of penetration of drug 3. Alteration of drug's target site 4. Rapid ejection of the drug Efflux Pumps • Modification of the antibiotic • Modification of the target structure

Transcription

A direct conversion of DNA to RNA by complementary base pairing where the units involved are closely related.

Six observed states of tRNA binding during its movement through the ribosome

A/T = cryo-EM A/A = X-ray + cryo-EM A/P = - P/P = X-ray + cryo-EM P/E = cryo-EM E/E = X-ray

Modification of the antibiotic

Active penicillin + Penicillinase = inactive penicillin E.g. the penicillinase enzyme disables the β-lactam ring making the drug redundant.

Factors involved in protein synthesis

DNA mRNA rRNA tRNA Amino acids

Recycling of EF-Tu

EF-Tu complexed to GTP escorts the aminoacyl tRNA to the ribosome. • The bound GTP is hydrolyzed as the correct tRNA is inserted, so EF-Tu complexed to GDP is released. • The EF-Tu/GDP complex is inactive and unable to bind another tRNA. In order for translation to continue, the active EF-Tu/GTP complex must be regenerated by another factor. • EF-Ts, which stimulates the exchange of the bound GDP for free GTP

Recycling of the ribosomes

Once the nascent protein is released in termination, Ribosome Recycling Factor and EF-G (eEF2) function to release mRNA and tRNAs from ribosomes. • The ribosomes then dissociate back into a small and a large subunit • IF3 (eIF3) then replaces the deacylated tRNA releasing the mRNA. • All translational components are now free for additional rounds of translation

Chloramphenicol

One of the first antibiotics to be used clinically Very cheap therefore tends to be used a lot in developing countries However have been pulled from use in the west due to a rare but lethal side effect known as aplastic anemia.

initiation of translation

The initiation of protein synthesis begins with the formation of an initiation complex. In E. coli, this complex involves the small 30S ribosome, the mRNA template, three initiation factors that help the ribosome assemble correctly, guanosine triphosphate (GTP) that acts as an energy source, and a special initiator tRNA carrying N-formyl-methionine (fMet-tRNAfMet) (Figure 1). The initiator tRNA interacts with the start codon AUG of the mRNA and carries a formylated methionine (fMet). Because of its involvement in initiation, fMet is inserted at the beginning (N terminus) of every polypeptide chain synthesized by E. coli. In E. coli mRNA, a leader sequence upstream of the first AUG codon, called the Shine-Dalgarno sequence (also known as the ribosomal binding site AGGAGG), interacts through complementary base pairing with the rRNA molecules that compose the ribosome. This interaction anchors the 30S ribosomal subunit at the correct location on the mRNA template. At this point, the 50S ribosomal subunit then binds to the initiation complex, forming an intact ribosome. In eukaryotes, initiation complex formation is similar, with the following differences: The initiator tRNA is a different specialized tRNA carrying methionine, called Met-tRNAi Instead of binding to the mRNA at the Shine-Dalgarno sequence, the eukaryotic initiation complex recognizes the 5′ cap of the eukaryotic mRNA, then tracks along the mRNA in the 5′ to 3′ direction until the AUG start codon is recognized. At this point, the 60S subunit binds to the complex of Met-tRNAi, mRNA, and the 40S subunit.

How do antibiotics, which affect protein synthesis, function?

These antibiotics exploit the structural and functional differences between bacterial and eukaryotic ribosomes. • They interact preferentially with bacterial ribosomes, interfering with their function . • As such humans can take high doses of these compounds without undue toxicity. • Many antibiotics lodge in pockets of rRNA and simply interfere with the smooth operation of the ribosome.

The peptidyl-transferase centre

an enzyme at the entrance of the peptide exit tunnel, catalyzes the reaction that forms peptide bonds between amino acids • Peptide bond formation is catalyzed by activity of the rRNA from the large subunit . • In this aspect the ribosome is said to act as a ribozyme (enzymatic ribosome).

Flow of Biological Information

draw the central dogma

Overview of protein synthesis

mRNA carrying our gene of interest • Aminoacylated tRNA's ready to make protein The Ribosome

Drug Discovery Process

1. target selection 2. lead discovery 3. medicinal chemistry 4. in vitro studies 5. in vivo studies 6. human trials • Long, complex, multi disciplinary process (12-15 years). • Very expensive. • High rates of attrition.

Where do antibiotics come from?

• Many of the most effective antibiotics used in modern medicine are compounds made by fungi. • Fungi and bacteria compete for many of the same environmental niches'. • As such fungi have evolved the ability to produce potent bacterial inhibitors.

Translation

• The translation of the mRNA codons into amino acid sequences leads to the synthesis of polypeptides, which then fold and/or aggregate to form functional molecules called proteins. • Proteins are the active participants in cell structure and function. • They are the "work horses" of the cell • The main function of the genetic material is, therefore, to encode the production of cellular proteins in the correct cell, at the proper time, and in suitable amounts

Prokaryotic and Eukaryotic Ribosomes

Although there is a large degree of similarity between prokaryotic and eukaryotic ribosomes, the differences which do exist between them can be exploited medically through the use of antibiotics. • Many antibiotics function by inhibiting protein synthesis, but these must specifically inhibit bacterial protein synthesis while not effecting human cell protein synthesis.

Elongation Factor Tu/1α

Aminoacyl tRNA's are delivered to the Ribosome by EFTu/1α. • A ternary complex is formed between the aminoacyltRNA, the EF and GTP to bring the incoming amino acid to the ribosome

tRNA visualization in the ribosome

Conformation of tRNA in the P/E hybrid state. (A) Movement of P/E tRNA and mRNA towards the E site when compared to P/P tRNA and mRNA. The direction of view is shown to the right. (B) View of mRNA and P/E tRNA interactions with the 30S subunit P site and 50S subunit E site. Residues that contact mRNA (gold) and P/E tRNA (red) are shown. Colors for the ribosome, mRNA and tRNA as in Fig. 1. (C) View of the P/E tRNA ASL/D stem junction (orange). P/P tRNA (grey) is shown for comparison, with an arrow indicating the widening of the helix major groove. (D) Comparison of ASL/D stem junctions between P/E tRNA (orange), P/P tRNA (grey), and A/T tRNA (purple). A/T tRNA structure is a homology model adapted from (12, 21). The bending angle for the A/T to P/E conformational change (70°) is shown.

Resistance to tetracyclines

Bacteria have developed resistance to tetracyclines by modifying their own ribosomes so that tetracycline can no longer bind. • This modification still allows the ribosomes to carry out protein synthesis effectively. • The protein which mediates this modification is closely related to, and probably evolved from, EF-G. • EF-G provides the energy for the mRNA and tRNA to translocate through the ribosome. • Tetracycline has been on the market since the late 1940's and has been one of the most widely used of all antibiotics. • In recent years its popularity has declined due to so many bacteria becoming resistant to it. • As this resistance began to appear new forms of tetracycline were used, such as doxycycline. • However today many bacteria have become resistant to all tetracyclines. • Despite this wide spread resistance tetracyclines are still very effective against two very serious diseases: Anthrax and Lymes disease

Mupirocin

Bactroban Topical Antibiotic The Exception is Mupirocin which inhibits the activity of the bacterial aminoacyl-tRNA synthatase. • However the overlap with human aa-tRNA synthatase showed this antibiotic to be toxic for internal use. • It is therefore primarily used as a topical antibiotic cream against antibiotic-resistant staphylococcus aureus strains such as MRSA

Why is the ribosome so big

Because it has a big substrate Fundamental mechanisms: • Aminoacyl-tRNA selection (30S) • Catalysis of peptide bond formation (50S) • Translocation (30S & 50S) • Maintenance of the translational reading frame (30S)

Mechanisms of Antibiotic Action

Cell Wall biosynthesis inhibition: Cephalosporins, glycopeptides and β-lactams (penicillins) Inhibition of DNA repair and replication: Fluoroquinolones Inhibition of protein biosynthesis: Tetracyclins, macrolides, aminoglycosides and oxazolidinones

Rare codon involvement in protein folding

Clustered codons that pair to low-abundance tRNA isoacceptors can form slow-translating regions in the mRNA and cause transient ribosomal arrest. We report that folding efficiency of the Escherichia coli multidomain protein SufI can be severely perturbed by alterations in ribosome-mediated translational attenuation. Such alterations were achieved by global acceleration of the translation rate with tRNA excess in vitro or by synonymous substitutions to codons with highly abundant tRNAs both in vitro and in vivo. Conversely, the global slow-down of the translation rate modulated by low temperature suppresses the deleterious effect of the altered translational attenuation pattern. We propose that local discontinuous translation temporally separates the translation of segments of the peptide chain and actively coordinates their co-translational folding.

Elongation in eukaryotes

Elongation depends on eukaryotic elongation factors. At the end of the initiation step, the mRNA is positioned so that the next codon can be translated during the elongation stage of protein synthesis. The initiator tRNA occupies the P site in the ribosome, and the A site is ready to receive an aminoacyl-tRNA. During chain elongation, each additional amino acid is added to the nascent polypeptide chain in a three-step microcycle. The steps in this microcycle are (1) positioning the correct aminoacyl-tRNA in the A site of the ribosome, (2) forming the peptide bond and (3) shifting the mRNA by one codon relative to the ribosome. Unlike bacteria, in which translation initiation occurs as soon as the 5' end of an mRNA is synthesized, in eukaryotes such tight coupling between transcription and translation is not possible because transcription and translation are carried out in separate compartments of the cell (the nucleus and cytoplasm). Eukaryotic mRNA precursors must be processed in the nucleus (e.g., capping, polyadenylation, splicing) before they are exported to the cytoplasm for translation. Translation can also be affected by ribosomal pausing, which can trigger endonucleolytic attack of the mRNA, a process termed mRNA no-go decay. Ribosomal pausing also aids co-translational folding of the nascent polypeptide on the ribosome, and delays protein translation while it is encoding mRNA. This can trigger ribosomal frameshifting

Macrolides

Erythromycin and other macrolides are widely used as they have few side effects. • Macrolides work by binding to the large subunit of the ribosome, where they prevent elongation of bacterial proteins. • Until the crystal structure of the ribosome was published this is all we knew. • Now we have crystalographic data of ribosomes with these antibiotics bound and we can clearly see that they bind to the inside walls of the ribosome exit tunnel. • These antibiotics create a physical obstruction to the elongation of the nascent chain so it can no longer leave the ribosome.

Eukaryotic Protein Synthesis

Eukaryotic translation is the biological process by which messenger RNA is translated into proteins in eukaryotes. It consists of four phases: initiation, elongation, termination, and recyclin Proteins are synthesized stepwise by the polymerization of amino acids in a unidirectional manner, beginning at the N-terminus and ending at the C-terminus. The amino acids are linked by the formation of peptide bonds, and the resulting polypeptide chain contains one of 20 different amino acids at each position. For protein synthesis, a messenger RNA (mRNA) molecule copied from DNA provides the instruction for the synthesis of a specific protein. The information encoded in the sequence of bases in the mRNA is translated by transfer RNA (tRNA) molecules that bind to the mRNA at one end, and carry specific amino acids at the other end. The synthesis of the growing polypeptide chain is carried out on ribosomes, that contain RNA and associated proteins. Additional specific protein factors aid in the initiation, elongation and termination of protein synthesis. Genetic information is encoded as a series of three bases, or triplets, in the mRNA. The 64 triplets and the amino acids they specify are called the genetic code. In most organisms three (and sometimes two) of the triplets signal chain termination

Structural properties of rRNA

For many years it was thought that the rRNAs in the ribosome served merely as a scaffold on which to hang the ribosomal proteins. • It was proposed that the proteins did all of the important work in the ribosome, such as catalyzing the formation of peptide bonds and moving the tRNAs and mRNA along during protein synthesis. • However, it is now clear that the rRNAs play an active role in protein synthesis and are not merely the frame on which the ribosome is built. • As more detailed information about the 3D structure of the ribosome becomes available a better understanding of what the rRNAs do and how they do it is becoming apparent.

hybrid state t-rna model

Here, the acceptor ends of the A- and P-site tRNAs move into the P and E sites of the large subunit, while the anticodon stem-loops of the tRNAs remain in the A and P sites of the small subunit. This configuration results in occupation by the tRNAs of the A/P and P/E hybrid states. Cryo-EM reconstructions indeed revealed structural evidence for a position of tRNA in the P/E hybrid state (11, 13, 18, 23); however, the A/P hybrid state has thus far eluded structural analysis using cryo-EM or x-ray crystallography. The initial report of hybrid-state tRNAs by Moazed and Noller (26) suggested that tRNAs occupy the hybrid state immediately after peptide bond formation, but structures representing the PRE ribosome determined by cryo-EM (13) and x-ray crystallography (32-35) showed that in the absence of elongation factors, the tRNAs occupy the classic A/A, P/P, and E/E sites exclusively. Potential explanations for this discrepancy are that the tRNAs are sampling conformations between classic and hybrid states in the PRE ribosome (36), or that the buffer used in the various studies stabilizes one state of binding over the other (i.e., classic over hybrid) the binding of EF-G/eEF2 in the GTP form stabilizes the ratcheted conformation, and with it, the hybrid-state tRNAs

Summary of antibiotic use in protein synthesis

In general a broad spectrum of macrolides are usually used to treat bacterial infections. • Failing this newer antibiotics such as Synercid and Linezolid are then used as a last attempt against stubbornly resistant bacteria. • Bacteria have however proven themselves to be just as smart as the drug companies by continually finding ways to resist these antibiotic treatments. • How long will it be before they develop resistance to these new types of antibiotic.

Ribosome Ratcheting

In the elongation cycle of translation, translocation is the process that advances the mRNA-tRNA moiety on the ribosome, to allow the next codon to move into the decoding center. New results obtained by cryoelectron microscopy, interpreted in the light of x-ray structures and kinetic data, allow us to develop a model of the molecular events during translocation

steps

In the first step of aminoacyltRNA synthesis, ATP and the appropriate amino acid form a high energy aminoacyl adenylate intermediate. Inorganic pyrophosphate is released and subsequently broken down to free phosphate by the enzyme inorganic pyrophosphatase. The aminoacyl adenylate is a "high-energy" intermediate, and in the second step, the transfer of amino acids to the acceptor end of tRNA occurs without any further input of ATP.

Scientists develop molecule that reverses antibiotic resistance

Infections which were previously easily treatable have grown immune to antibiotics •The news comes after a woman died last year from an infection that was resistant to every kind of antibiotic. These superbugs have been deemed a "fundamental threat" by the United Nations and it is predicted they will kill 300 million people by 2050

Initiation of Translation - Prokaryotes

Initiation of translation in prokaryotes involves the assembly of the components of the translation system which are: - the two ribosomal subunits - the mRNA to be translated - the first (formyl) aminoacyl tRNA - GTP (as a source of energy), - three initiation factors (IF1, IF2 and IF3) which help the assembly of the initiation complex. Initiation of translation begins with the 50S and 30S ribosomal subunits dissociated. IF1 (initiation factor 1) blocks the A site to ensure that the fMet-tRNA can bind only to the P site and that no other aminoacyl-tRNA can bind in the A site during initiation. • IF3 blocks the E site and prevents the two subunits from associating . • IF2 is a small GTPase which binds fmet-tRNAfMet and helps its binding with the small ribosomal subunit.

Inosine

Inosine is commonly found in tRNAs and is essential for proper translation of the genetic code in wobble base pairs. it most closely resembles guanine and it can base pair with either A, U, or C which gives the tRNA's more flexibility. Some nucleotides of the tRNA undergo post-transcriptional modification. • The best example of this is Inosine which results from the deamination of adenosine. • Inosine is commonly found at the wobble position where it facilitates the recognition of the appropriate mRNA codon by the tRNA molecule

Due to the fierce nature of such antibiotics many side effects are also seen

Joint aches (arthralgia) • muscle aches (myalgia) • Nausea, diarrhea or vomiting • Rash or itching • Headache • Hyperbilirubinemia Synercid compounds also bind within the ribosome exit tunnel of the large subunit. • As two compounds are used it is hoped that its effect is more pronounced and less susceptible to resistance. Binding of antibiotics to the ribosome exit tunnel has been a very effective way to kill bacteria. • This is clearly shown by the overlapping binding sites of many antibiotics. • However this leads to a problem in cross-resistance selection.

Other antibiotics which bind to the ribosome exit tunnel

Lincosiamides which do not resemble macrolides structurally have also been shown to bind to the ribosome tunnel. • It has been shown that the binding sites for these antibiotics, although not the same, overlaps with that of the macrolides creating a similar obstruction. • The problem with these overlapping sites is that some bacteria have developed and enzyme which methylates a key residue on the 23S rRNA of the large subunit. • This small change in structure is enough to simultaneously confer resistance to all these classes of antibiotic by reducing their binding to ribosomes

Rare Codons - codon optimisation

Most amino acids are coded by multiple codons, and hence have multiple tRNAs. • Not all codons are created equal, some codons are found much less frequently than others that represent the same amino acid. • The tRNA associated with these "rare codons" are less abundant than other tRNAs. • When a ribosome hits a rare codon, it has to pause while it waits to encounter a loaded tRNA. • Can be optimised to increase protein expression. • Can be potentially useful

Peptide bond synthesis

Next a peptide bond will form between the amino acid subunits in the P- and A-sites to form a polypeptide chain. • The linkage is fromed between the carboxyl group of the amino acid at the P-site and the amide group of the amino acid at the A-site As a result the methionine shifts over to the A site, with the original amino acid on the A site as the lowest link in the chain. The tRNA in the A site becomes peptidyl RNA. • The ribosome engages in a process called translocation.

Oxazolidones

Oxazolidones such as linezolid were first synthesized in the late 1980's and are only now becoming available commercially. • These antibiotics are seen as a new hope in the field of antibiotics as they act at an earlier stage in protein synthesis than almost any other antibiotic, except streptomycin. • They act by by stopping the 30S and 50S subunits of the ribosome from binding together. • In contrast to synercid, oxazolidones, are unlikley to be targets for the development of cross-resistance selected by other classes of antibiotic. Linezolid • Linezolid binds on the 23S portion of the 50S subunit close to the peptidyl transferase center. • This then stops the interaction with the 30S subunit

Streptogramins: Synercid

Protein Synthesis Inhibitor (Ribosome 50s inhibitor) Streptogramins are effective in the treatment of Vancomycin-resistant Staphylococcus aureus (VRSA) and Vancomycin-resistant enterococcus (VRE), two of the most rapidly-growing strains of multi-drug resistant bacteria. • Synercid is a combination of to compounds quinupristin and dalfopristin. • These are produced by streptomyces pristinaspiralis and are very new to the clinical drug market. • Synercid is marketed as a replacement for vancomycin, which until recently was the only treatment for methacillin resistant staphylococcus aureus

Hybrid States of tRNA Translocation

Protein synthesis on ribosomes, that is, the translation of the nucleotide sequence of mRNA into the amino acid sequence of proteins, is a cyclic process. In each round of elongation, two tRNA molecules together with the mRNA move through the ribosome in a multistep process called translocation. Thus, peptidyl-tRNA moves from the A site of the ribosome, where decoding takes place, to the P site, where the peptidyl-tRNA is placed before transferring the nascent peptide onto the aminoacyl-tRNA that enters the A site in the next round of elongation. In parallel, deacylated tRNA moves from the P site to the E (exit) site before dissociating from the ribosome. Translocation is arguably the most complex step of ribosomal protein synthesis and involves large-scale conformational changes of the ribosome. The physiological reaction is promoted by an elongation factor (EF-G in bacteria or eEF2 in eukaryotes), which hydrolyzes GTP in the process.

eukaryotic ribosomes

The small ribosomal subunit is made up of 18S rRNA and approximately thirty-three proteins . The large ribosomal subunit contains three rRNAs - 5S, 5.8S and 28S - and approximately forty-nine proteins.

Overcoming Antibiotics

Staphylococci bacteria, which have been labelled with a green fluorescent protein, express a resistance gene for the antibiotic chloramphenicol. Black Streptococcus pneumoniae bacteria do not have the resistance gene. In a medium containing the antibiotic, the green cells begin to grow and divide whereas the non-resistant black cells don't. After a time, individual black cells begin to divide and they even outgrow their green companions

The genetic code selects its amino acid through a two step process

Step 1 - tRNA selection of amino acid • Step 2 - aa-tRNA selection by mRNA codon look for diagram in lecture 2

Tetracyclines

Tetracyclines get their name from their structure as they consist of 4 fused rings. Tetracyclines, like streptomycin, bind the 30S subunit of the ribosome. They act by distorting the structure of the A -site of the small subunit so that incoming aminoacyl -tRNA's can no longer interact properly with the mRNA.

The Ribosome - prokaryote

The active site is formed when the two subunits of the ribosome come together encapsulating the mRNA. • The ribosome is made up of two subunits. Each of the subunits is made up of both protein and rRNA. The small ribosomal subunit is made up of one rRNA and twenty-one proteins The large ribosomal subunit contains two rRNAs -5S and 23Sand thirty-four proteins.

Editing

The correct amino acid has the highest affinity for the active site pocket of the synthetase. Errors are excluded in two ways: Matching the correct amino acid with the correct tRNA is the job of Aminoacyl tRNA Synthetase enzymes. They have to be very specific to get the right tRNA and the right amino acid, those are 2 separate jobs. The amino acid binds to the activation site, and the tRNA interaction is at several different sites. The amino acid will be attached to the 3' end of the tRNA which is always the same, CCA. You might expect that the enzyme would only need to look at the anticodon, but in fact there are "clues" in other places. The yellow areas show where one aminoacyl tRNA synthetase binds its tRNA. After the amino acid is attached to the 3' end of the tRNA it is "double checked" which is necessary because some amino acids have very similar structures to others, like Valine and Isoleucine. look at diagram lecture 2

Initiation in Eukaryotes

The eukaryotic initiation 4 complex binds to the cap structure at the 5' end of the mRNA. • Eukaryotic initiation factor 3 (eIF3) is associated with the small ribosomal subunit, and plays a role in keeping the large ribosomal subunit from prematurely binding . • eIF3 also interacts with the eIF4 complex. • The Poly(A)-binding protein (PABP), also associates with the eIF4 complex. • PABP binds the poly-A tail of most eukaryotic mRNA molecules circularization of the mRNA during translation. • The complex formed is known as the 48S pre-initiation complex This pre-initiation complex moves along the mRNA chain towards its 3'-end, scanning for the 'start' codon (typically AUG) on the mRNA, which indicates where the mRNA will begin coding for the protein. • As there is no Shine-Dalgarno sequence, the ribosome begins translation at a AUG that is located within the Kozak consensus sequence, usually CAAAAUG. • The Met-charged initiator tRNA is brought to the P-site of the small ribosomal subunit by eukaryotic Initiation Factor 2 (eIF2). eF2 hydrolyzes GTP, and signals for the dissociation of several factors from the small ribosomal subunit which results in the association of the large subunit (or the 60S subunit). • The complete ribosome (80S) then commences translation elongation, during which the sequence between the 'start' and 'stop' codons is translated from mRNA into an amino acid sequence -- thus a protein is synthesized. With both prokaryotic and eukaryotic systems we have seen how the initiation complexes form. • In both cases a fully formed ribosome is bound to the mRNA with an initiator Met-tRNA at the P-site. • The mRNA is positioned at the first codon of the open reading frame

Gene regulation by premature termination

The genetic code is said to be redundant in that the same amino acid residue can be encoded by multiple, so-called synonymous, codons. If all properties of synonymous codons were entirely equivalent, one would expect that they would be equally distributed along protein coding sequences. However, many studies over the last three decades have demonstrated that their distribution is not entirely random. It has been postulated that certain codons may be translated by the ribosome faster than others and thus their non-random distribution dictates how fast the ribosome moves along particular segments of the mRNA. The reasons behind such segmental variability in the rates of protein synthesis, and thus polypeptide emergence from the ribosome, have been explored by theoretical and experimental approaches. Predictions of the relative rates at which particular codons are translated and their impact on the nascent chain have not arrived at unequivocal conclusions. This is probably due, at least in part, to variation in the basis for classification of codons as "fast" or "slow", as well as variability in the number and types of genes and proteins analyzed. Recent methodological advances have allowed nucleotide-resolution studies of ribosome residency times in entire transcriptomes, which confirm the non-uniform movement of ribosomes along mRNAs and shed light on the actual determinants of rate control. Moreover, experiments have begun to emerge that systematically examine the influence of variations in ribosomal movement and the fate of the emerging polypeptide chain.

Shine-Dalgarno sequence

The prokaryotic ribosome-binding site on mRNA, found 10 nucleotides 5' to the start codon. The Shine-Dalgarno sequence is a purine-rich sequence (GAGGGG) found in the initiator region of prokaryotic mRNA. It is located about 10 nucleotides upstream the initiator codon AUG. • This sequence binds to a complementary region near the 3'- end of the 16S rRNA. • This region therefore has influence on where the translation process starts. Protein synthesis actually begins with the interaction of the Shine-Dalgarno sequence of the mRNA with the rRNA of the ribosome. The 3' end of the 16S rRNA of the small 30S ribosomal subunit recognizes the Shine-Dalgarno sequence on the mRNA. • The Shine-Dalgarno sequence helps to correctly position the ribosome onto the mRNA so that the P site is directly on the AUG initiation codon. IF3 helps to position fMet-tRNAfMet into the P site, such that fMet-tRNAfMet interacts via base pairing with the mRNA initiation codon (AUG). Initiation ends as the large ribosomal subunit joins the complex causing the dissociation of initiation factors.

Path of the newly synthesised protein

The ribosome tunnel is approximately 100 Å long stretching from the peptidyl-transferase centre to the exit at the base of the ribosome. Its diameter is between 10 and 20 Å starting on the smaller side and becoming wider as it nears the exit.

Structure/Function Relationship

The structure of a ribosome reflects its function of bringing mRNA together with charged tRNAs. • Each ribosome has three binding sites for tRNA: - The A site (aminoacyl) - holds the tRNA carrying the next amino acid to be added to the chain - The P site (peptidyl) - holds the tRNA carrying the growing polypeptide - The E site (exit) - discharged tRNA's leave the ribosome through this site.

Movement of tRNA through the Ribosome

The tRNA binds to a groove at the bottom of the mRNA tunnel. • After each amino acid is added to the growing protein, the tRNAs must be moved from one site to the next. • The mRNA must also be moved over one codon so that the next amino acid coded for by the mRNA can be added to the protein. • During protein synthesis all three sites are occupied

Termination

The termination of translation occurs when a nonsense codon (UAA, UAG, or UGA) is encountered for which there is no complementary tRNA. On aligning with the A site, these nonsense codons are recognized by release factors in prokaryotes and eukaryotes that result in the P-site amino acid detaching from its tRNA, releasing the newly made polypeptide. The small and large ribosomal subunits dissociate from the mRNA and from each other; they are recruited almost immediately into another translation init iation complex. draw the differences

Genetic Code

The translation of mRNA to protein is mediated by the "Genetic code". • This code was deciphered in 1961 by Marshall Nirenberg at the US National Institutes of Health, and who received the Nobel prize for his work. • The sequence of nucleotides in the mRNA is read in consecutive groups of three. • As there are 4 nucleotide bases there are therefore 64 possible combinations of triplets.

initiatiion summary

Translation initiation: Initiation factorsProkaryotes require the use of three initiationfactors: IF1, IF2, and IF3, for translation.IF1 associates with the 30S ribosomal subunit in theA site and prevents an aminoacyl-tRNA from entering. Itmodulates IF2 binding to the ribosome by increasing itsaffinity. It may also prevent the 50S subunit from binding,stopping the formation of the 70S subunit. It also containsa β-domain fold common for nucleic acid binding proteins. 7. Translation initiation: Initiation factorsIF2 binds to an initiator tRNA and controls the entryof tRNA onto the ribosome. IF2, bound to GTP, binds to the30S P site. After associating with the 30S subunit, fMettRNAf binds to the IF2, then IF2 transfers the tRNA intothe partial P site. When the 50S subunit joins, it hydrolyzesGTP to GDP and Pi, causing a conformational change in theIF2 that causes IF2 to release and allow the 70S subunit toform. 8. Translation initiation: Initiation factorsIF3 is not universally found in all bacterial speciesbut in E. coli it is required for the 30S subunit to bind tothe initiation site in mRNA. In addition, it has severalother jobs including the stabilization of free 30Ssubunits, enables 30S subunits to bind to mRNA andchecks for accuracy against the first aminoacyl-tRNA. Italso allows for rapid codon-anticodon pairing for theinitiator tRNA to bind quickly to. IF3 is required by thesmall subunit to form initiation complexes, but has to bereleased to allow the 50S subunit to bind.

Addition of the 3rd amino acid

With the A site open, the next appropriate aminoacyl tRNA can bind there and the same reaction takes place, yielding a three-amino acid peptide chain. This process repeats, creating a polypeptide chain in the P site of the ribosome. • Eukaryotic ribosomes work at a rate of 2aa/sec. • Prokaryotic ribosomes work at a rate of 20aa/sec.

editing

Translation of mRNA into proteins involves two key steps of the decoding process. Aminoacyl tRNA synthetases (aaRSs) first translate the genetic code into amino acids and then attach the correct amino acids to their cognate tRNAs. The charged tRNAs are subsequently brought to the ribosomes and positioned on the mRNA, allowing completion of protein synthesis. The aminoacylation reaction itself proceeds in two stages: (i) activation of the amino acid by ATP, leading to synthesis of an amino acid adenylate; and (ii) charging of the amino acid at the CCA end of the cognate tRNA. Two classes of enzymes with distinct active site structures are responsible for the accuracy of the reaction (3). Editing can occur either before (pretransfer editing) or after (posttransfer editing) a misactivated amino acid is attached to tRNA. The first experimental evidence for these mechanisms as well as their theoretical analysis was established in the 1970s (see ref. 4 for a good review of the problem). The first demonstration of posttransfer editing was provided by Eldred and Schimmel (5) and by Yarus (6), who found that IleRS and PheRS catalyzed the hydrolysis of misacylated Val-tRNAIle and Ile-tRNAPhe, respectively. Later Fersht demonstrated the existence of pretransfer editing by fast-kinetic studies of IleRS (7). Today approximately half of all aaRSs have been shown to rely on editing to efficiently discriminate cognate from structurally similar noncognate amino acids.

Antibiotics which inhibit protein synthesis

With one exception, all currently available antibiotics which inhibit bacterial protein synthesis do so by binding the ribosome Aminoglycosides = Streptomycin/ Amikacin/ Neomycin Tetracyclines= Tetracycline /Doxycycline/ Oxytetracycline/ Demeclocycline/ Minocycline Macrolides =Erythromycin/ Azithromycin Lincosamides =Clindamycin Streptogramin = Quinupriston + Dalfopriston (Synercid) Oxazolidone = Linezolid Mupirocin= Mupirocin

Adaptor Hypothesis

a hypothesis that proposes a tRNA has two functions: recognizing a three-base codon sequence in mRNA and carrying an amino acid that is specific for that codon the position of an amino acid within a polypeptide is determined by the binding between the mRNA and an adaptor molecule carrying a specific amino acid mRNA codons are not directly recognised by amino acids. • Adaptor molecules which recognize both the mRNA and the amino acid bring the two together. • In the 1950s Francis Crick proposed the adaptor hypothesis, which hypothesized that a molecule called transfer RNA (tRNA) played a direct role in the recognition of codons in the mRNA. In particular, the hypothesis proposed that tRNA has two functions: 1. Recognizing a 3-base codon in mRNA 2. Carrying an amino acid that is specific for that codon to the translation machinery

elongation (translation)

begins by the appropriate aminoacyl-tRNA binding to the codon in the A site of the ribosome. In prokaryotes and eukaryotes, the basics of elongation of translation are the same. In E. coli, the binding of the 50S ribosomal subunit to produce the intact ribosome forms three functionally important ribosomal sites: The A (aminoacyl) site binds incoming charged aminoacyl tRNAs. The P (peptidyl) site binds charged tRNAs carrying amino acids that have formed peptide bonds with the growing polypeptide chain but have not yet dissociated from their corresponding tRNA. The E (exit) site releases dissociated tRNAs so that they can be recharged with free amino acids. There is one notable exception to this assembly line of tRNAs: During initiation complex formation, bacterial fMet−tRNAfMet or eukaryotic Met-tRNAi enters the P site directly without first entering the A site, providing a free A site ready to accept the tRNA corresponding to the first codon after the AUG. Elongation proceeds with single-codon movements of the ribosome each called a translocation event. During each translocation event, the charged tRNAs enter at the A site, then shift to the P site, and then finally to the E site for removal. Ribosomal movements, or steps, are induced by conformational changes that advance the ribosome by three bases in the 3′ direction. Peptide bonds form between the amino group of the amino acid attached to the A-site tRNA and the carboxyl group of the amino acid attached to the P-site tRNA. The formation of each peptide bond is catalyzed by peptidyl transferase, an RNA-based ribozyme that is integrated into the 50S ribosomal subunit. The amino acid bound to the P-site tRNA is also linked to the growing polypeptide chain. As the ribosome steps across the mRNA, the former P-site tRNA enters the E site, detaches from the amino acid, and is expelled. Several of the steps during elongation, including binding of a charged aminoacyl tRNA to the A site and translocation, require energy derived from GTP hydrolysis, which is catalyzed by specific elongation factors. Amazingly, the E. coli translation apparatus takes only 0.05 seconds to add each amino acid, meaning that a 200 amino-acid protein can be translated in just 10 seconds.

Aminoacylation of tRNA

formation of aminoacyl adenylate which remains bound to the active site, aminoacyl group is transferred to tRNA At the opposite end of the tRNA amino acids are loaded onto the correct tRNA as specified by the anticodon in a process known as aminoacylation. Aminoacylation is a two-step process, catalyzed by a set of enzymes known as aminoacyl-tRNA synthetases. • Most cells have twenty aminoacyl-tRNA synthetases, one per amino acid in the genetic code. • Bacteria have fewer than this hence in some cases a synthetase enzyme will couple more than one amino acid.

Modification of the target structure

interaction to no interaction The activity of vancomycin is reduced x1000 fold by a single amino acid change on the target which results in the loss of a key H-bond

The Ternary Complex

protein complex containing three different molecules that are bound together A ternary complex is formed between the aminoacyl-tRNA, EF-Tu and GTP to bring the incoming amino acid to the ribosome. • Binding of this complex at the A-site hydrolysis GTP and releases EF-Tu with GDP bound.

tRNA Structure

secondary structure, cloverleaf form, anticodon end is opposite 3' aminoacyl end. All tRNAs, both eukaryotic and prokaryotic, have CCA at 3' end along w/ a high percentage of chemically modified bases. The AA is covalently bound to the 3' end of tRNA. tRNA molecules can be drawn schematically as a clover leaf design to show their 4 domains. • Their key features are - An adaptor stem = Amino acid binding site. - 3' single stranded region (anticodon) = three consecutive nucleotides that pair with the complimentary mRNA codon. • The correctly folded 3D structure of tRNA looks more like and L-shape, where the anticodon and the amino acid binding site are well separated. tRNA's bring amino acids into close proximity so peptide bonds can be formed

Wobble Hypothesis

the hypothesis that some tRNA molecules can pair with more than one mRNA codon, tolerating some variations in the third base, as long as the first and second bases are correctly matched In 1966 Francis Crick proposed the wobble hypothesis. • When a codon is recognised by an anticodon the first 2 positions pair strictly to the A-U / G-C rule. • The third position can wobble or move a bit, thus tolerating certain types of mistakes.

Tunnel Dynamics - : Implications of helix formation within the ribosomal exit tunne

the transmembrane helix maintains a helical conformation during its passage throughout the entire ribosomal tunnel. Although we observed no helix formation in the middle of the tunnel (that is, directly following the constriction (Fig. 4a)), the observed discrepancy can be related to the use of a hydrophobic transmembrane helix13 rather than the hydrophilic helix used here. In any case, the ability to form a helix near to the tunnel exit site but not in the middle region of the tunnel is consistent with the zones of secondary-structure formation identified previously Schematic representation of a cross-section of the 80S-helix1 RNC showing the regions where helix and tertiary structure formation are observe

How do 64 different codons produce 20 different amino acids?

to pick the right amino acids, a ribosome takes the nucleotides in sets of three to encode for the 20 amino acids. What this means is that every three base pairs in the DNA chain encodes for one amino acid in an enzyme. Three nucleotides in a row on a DNA strand is therefore referred to as a codon. Because DNA consists of four different bases, and because there are three bases in a codon, and because 4 * 4 * 4 = 64, there are 64 possible patterns for a codon. Since there are only 20 possible amino acids, this means that there is some redundancy -- several different codons can encode for the same amino acid. In principle an RNA molecule can be translated in anyone of 3 different reading frames. • However only one encodes the protein; this is determined by upstream promotors and ribosome binding domains. Although multiple codons are seen for single amino acids, there are usually dominant codons

Resistance to aminoglycosides

transferase enzymes that inactivate the drug by acetylation, phosphorylation, or adenylation. Mutation of the interacting ribosomal proteins can lead to resistance from the specified antibiotic. • Bacteria with mutated ribosomal proteins do not thrive well and are less infectious. • Bacteria have come up with a unique way to overcome this. • Many aminoglycoside resistant bacteria have acquired an enzyme which modifies the antibiotic by covalently attaching chemical groups to it. • This modification prevents the antibiotic binding to the ribosome, thus eliminating its ability to stop protein synthesis.

Efflux Pumps

transmembrane pump that removes antimicrobial drugs from a cell or from the periplasm Antibiotics (A) penetrate the cell and build up in concentration until the drug acts to kill the bacteria. • However many bacteria have evolved lipophilic proteins that begin to remove or "efflux pump out" the drug. • The concentration of the drug becomes too low to have any killing effect and the bacteria survive and continue to replicate

Aminoglycosides

• Aminoglycosides are among the most widely used antibiotics. • They were discovered by Selman Waxman in 1943 who then won the Nobel prize for his discovery in 1952. • Their name comes from the fact that the antibiotics of this family are composed of sugars with amino groups attached to them. • Streptomycin is the most commonly known aminoglycoside, it interferes at a very early stage in protein synthesis when the mRNA becomes associated with the small subunit of the ribosome. Streptomycin binds to a protein in the 30S subunit and acts by freezing the ribosome on the mRNA so that initiation can not continue. Other members of the aminoglycoside family such as amikacin, kanamycin and neomycin prevent later stages of protein synthesis such as the translocation of tRNA and mRNA. • They all act by binding to proteins on either the small or large subunit of the ribosome.

Translocation of the mRNA and the peptidyl tRNA

• EF-G-GTP then binds to the ribosome. • GTP is hydrolysed which promots the ribosome to move three nucleotides in the 3' prime direction along the mRNA. • In other words, the ribosome moves so that a new mRNA codon is accessible in the A-site. • At the same time the peptidyl tRNA is translocated from the A- to the P-site. • And the discharged tRNA moves from the P- to the E-site.