Objectives: Prof.Dr. H.D.El-Yassin

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1 Protein Synthesis and drugs that inhibit protein synthesis Objectives: 1. To understand the steps involved in the translation process that leads to protein synthesis 2. To understand and know about all the structures and molecules involved in protein synthesis 3. know about the drugs that inhibits protein synthesis and the mechanism of inhibition Prof.Dr. H.D.El-Yassin

2 Protein biosynthesis is the process in which cells build proteins. The term is sometimes used to refer only to protein translation, but more often it refers to a multi-step process, beginning with transcription and ending with proteintranslation Prof.Dr. H.D.El-Yassin

3 Ribosome Figure: Ribosome structure indicating small subunit (A) and large subunit (B). Side and front view.(1) Head. (2) Platform. (3) Base. (4) Ridge. (5) Central protuberance. (6) Back. (7) Stalk. (8) Front. A ribosome is an organelle composed of rrna (synthesized in the nucleolus) and ribosomal proteins. It translates mrna into a polypeptide chain (e.g., a protein). It can be thought of as a factory that builds a protein from a set of genetic instructions. Free ribosomes Free ribosomes occur in all cells. Free ribosomes usually produce proteins that are used in the cytosol or in the organelle they occur in. Membrane bound ribosomes When certain proteins are synthesized by a ribosome, it can become "membrane-bound", associated with the membrane of the nucleus and the rough endoplasmic reticulum (in eukaryotes only) for the time of synthesis Prof.Dr. H.D.El-Yassin

4 The ribosomal subunits of prokaryotes and eukaryotes are quite similar. However, prokaryotes use 70S ribosomes, each consisting of a (small) 30S and a (large) 50S subunit, whereas eukaryotes use 80S ribosomes, each consisting of a (small) 40S and a bound (large) 60S subunit.[the unit S means Svedberg units, a measure of the rate of sedimentation of a particle in a centrifuge, where the sedimentation rate is associated with the size of the particle]. Figure : Translation (1) of mrna by a ribosome (2) into a polypeptide chain (3). The mrna begins with a start codon (AUG) and ends with a stop codon (UAG). Translation (also called protein biosynthesis or polypeptide synthesis) is the second process in gene expression. In translation, messenger RNA is used as a template to produce a specific polypeptide according to the rules specified by the genetic code. Phases Translation proceeds in three phases: initiation, elongation, and termination (all describing the growth of the amino acid chain, or polypeptide that is the product of translation) Prof.Dr. H.D.El-Yassin

5 1. Initiation of translation involves the small ribosomal subunit binding to the 'start' codon on the mrna, which indicates where the mrna starts coding for the protein. This codon is most commonly an AUG. In eukaryotes amino acid encoded by the start codon is methionine. In bacteria, the protein starts instead with the modified amino acid N- formyl methionine (f-met). In f-met, the amino group has been blocked by a formyl group to form an amide, so this amino group can not form a peptide bond. This is not a problem because the f-met is at the amino terminus of the protein. 2. The large subunit then forms a complex with the small subunit, and elongation proceeds. A new activated trna enters the A site of the ribosome and base pairs with the mrna. The enzyme peptidyl transferase forms a peptide bond between the adjacent amino acids. As this happens, the amino acid on the P site leaves its trna and joins the trna at the A site. The ribosome then moves in relation to the mrna shifting the trna at the A site on to the P whilst releasing the empty trna, this process is known as translocation. 3. This procedure repeats until the ribosome encounters one of three possible stop codons, where translation is terminated. This stalls protein growth, and release factors, proteins which mimic trna, enter the A site and release the protein in to the cytoplasm. Synthesis of proteins can take place extremely quickly. This is aided by multiple ribosomes being able to attach themselves to one mrna chain, thus allowing multiple proteins to be constructed at once. An mrna chain with multiple ribosomes is called a polysome. Also, as prokaryotes have no nucleus, an mrna can be translated while it is still being transcribed. This is not possible in eukaryotes as translation occurs in the cytoplasm, whereas transcription occurs in the nucleus Prof.Dr. H.D.El-Yassin

6 Protein Synthesis in Eukaryotes A major difference between eukaryotes and prokaryotes is that, in a typical eukaryotic cell, protein synthesis takes place in the cytoplasm while transcription and RNA processing take place in the nucleus. In bacteria, these two processes can be coupled so that protein synthesis can start even before transcription has finished. INITIATION The cap-dependent translation initiation pathway Cap-dependent initiation is the major translation initiation pathway in eukaryotes eukaryotic mrnas are monocistronic, capped at the 5' end and polyadenylated at the 3' end ribosomes dissociate into 40S and 60S subunits Prof.Dr. H.D.El-Yassin

7 40S subunits locate the initiator AUG codon by scanning the mrna from the cap structure in the 3' direction for the first AUG codon at the AUG codon the 60S ribosomal subunit joins the 40S initiation complex to form an 80S ribosome competent for translation elongation: Fig.: Principle of cap-dependent translation initiation. AUU, stop codon; AAAn, poly(a) tract. A large number of proteins, the eukaryotic translation initiation factors (eif) catalyze individual steps in the pathway. ELONGATION: The elongation in eukaryotes is very similar to that in prokaryotes. TERMINATION: Mechanism in eukaryotes is similar to that in prokaryotes Prof.Dr. H.D.El-Yassin

8 Drugs that inhibits protein synthesis 1. Erythromycin Mechanism of Action Erythromycin inhibit protein synthesis by binding to the 23S rrna molecule (in the 50S subunit) of the bacterial ribosome blocking the exit of the growing peptide chain. (Humans do not have 50 S ribosomal subunits, but have ribosomes composed of 40 S and 60 S subunits). Certain resistant microorganisms with mutational changes in components of this subunit of the ribosome fail to bind the drug. The association between erythromycin and the ribosome is reversible and takes place only when the 50 S subunit is free from trna molecules bearing nascent peptide chains. The non ionized from of the drug is considerably more permeable to cells, and this probably explains the increased antimicrobial activity that is observed in alkaline ph. 2. Tetracyclines: Tetracyclines have the broadest spectrum of antimicrobial activity. Four fused 6-membered rings, as shown in the figure below, form the basic structure from which the various tetracyclines are made Mechanism of Action: Tetracyclines inhibit bacterial protein synthesis by blocking the attachment of the transfer RNA-amino acid to the ribosome. More precisely they are inhibitors of the codon-anticodon interaction. Tetracyclines can also inhibit protein synthesis in the host, but are less likely to reach the concentration required because eukaryotic cells do not have a tetracycline uptake mechanism Prof.Dr. H.D.El-Yassin

9 3. Streptomycin: Streptomycin binds to the 30S ribosome and changes its shape so that it and inhibits protein synthesis by causing a misreading of messenger RNA information. 4. Chloramphenicol: Chloromycetin is also a broad spectrum antibiotic that possesses activity similar to the tetracylines. At present, it is the only antibiotic prepared synthetically. It is reserved for treatment of serious infections because it is potentially highly toxic to bone marrow cells. It inhibits protein synthesis by attaching to the ribosome and interferes with the formation of peptide bonds between amino acids Prof.Dr. H.D.El-Yassin

10 Conclusion: 1. Proteins are produced by the process of translation which involves three main steps: a. Initiation involves formation of a complex containing the initial methionyltrna bound to the AUG start codon of the mrna and to the P site of the ribosome. b. Elongation of the polypeptide involves three steps: i. binding of an aminoacyl-trna to the A site on the ribosome where it base-pairs with the secondcodon on the mrna; ii. formation of a peptide bond between the first and second amino acids; and iii. (c) translocation, movement of the mrna relative to the ribosome, so that the third mrna codon moves into the A site. c. These three elongation steps are repeated until a termination codon aligns with the site on the ribosome where the next aminoacyl-trna would normally bind. 2. Protein synthesis occurs on ribosomes and is directed by mrna. mrna with multiple ribosomes is known as a polysome. 3. each step in the protein synthesis might me inhibited by a certain drug Prof.Dr. H.D.El-Yassin