Protein Synthesis Translation

In this lecture, we will be discussing the last step in protein synthesis: Translation. General Information After DNA is transcribed into an mRNA molecule “Transcription”, the mRNA is translated into amino acid sequences building up a protein “Translation”. Translation involves interactions between 3 types of RNA molecules: 1- tRNA, carrying the amino acids. 2- rRNA, as a part of ribosomes. 3- mRNA, as a template for translation. Note: The ribosome is made up of 60% rRNA and 40% protein by weight. tRNA Structure - tRNAs are small, non-coding, RNA molecules composed of, typically, 80 bases long. They have the same bases present in mRNA, in addition to inosine sometimes. - On the 3’ end of the tRNA, there is a consensus sequence of CCA. An amino acid binds covalently (ester bond) to the adenosine “A” of that sequence, catalyzed by an AminoacyltRNA synthetase producing a ‘charged/activated’ tRNA molecule. - For every amino acid, there is a special aminoacyl-tRNA synthetase. Thus, there are 20 different aminoacyl-tRNA synthetases. - Another region on the tRNA is called the anti-codon region, which is what determines which amino acid binds to the tRNA. It is complementary to the codon on the mRNA being translated binding anti-parallelly to it. Codons - Every three nucleotides in mRNA make up one codon which codes for a certain amino acid. Multiple codons, usually, code for one amino acid except for Methionine which is coded by only one codon. - In mRNA, we have 64 possible codons (43), as shown in this picture to the right. 2|P a ge The codons are not for memorization, the doctor said he will bring the table if we needed it, just learn the following: 1- AUG: It is a start codon which codes for Methionine. This implies that most peptide chains, typically, start with Methionine. 2- UAA, UAG, UGA: They are stop codons that do not code for any amino acid having no specific anti-codons, terminating the translation process. Therefore, we have 61 different tRNA molecules (64 - 3 stop codons) each with a specific anticodon. Some organisms have fewer than 45 tRNA molecules, therefore, one tRNA needs to pair with more than one codon (explained how later in wobble base pairing). Universality of Codons - Codons are usually universal, meaning that a code can be transcribed and translated into the same amino acids in many different organisms. - However, they are not always universal. This means that a codon can differ between prokaryotes and eukaryotes, the same organism can also contain 2 different genetic codes. For example, AUA in the mitochondrial genome codes for Methionine whereas in the genomic DNA it codes for Isoleucine. Wobble Base Pairing - The first two bases in a codon on the mRNA are usually what create the specificity for a certain amino acid-charged tRNA, forming strong bonds with the anticodon on the tRNA. - However, the 3rd base usually has a ‘relaxed’ bond with the anticodon of the tRNA (with the 1st base) making it degenerative/non-standard; meaning that it can avoid standard base-pairings. - If the 1st base on the anticodon is a C or an A, pairing is specific. However, if the 1st base is U or G, the pairing is less specific and more flexible and can be interchangeably recognized. If the 1st base is inositol, it can pair with A, C, or U. This degeneracy can be useful by: 1- Acting as a buffer against mutations. So that if there is a mutation in the wobble base, it won’t change the amino acid translated. 2- Our bodies have a limited amount of tRNAs and the wobble phenomenon decreases the required number of different tRNAs. Note: The amino acid attached to the tRNA is specified not only by the anticodon, but also by the identifier sequence (we are not required to know it in details yet). 3|P a ge Ribosomes Ribosomes are sites of protein synthesis in both prokaryotic and eukaryotic cells. E. coli contain about 20,000 ribosomes, which account, approximately, for 25% of the dry weight of the cell. While rapidly growing mammalian cells contain about 10 million ribosomes. They consist of proteins and rRNA. The function of the proteins is to maintain the structure, while the rRNA has enzymatic activity. Ribosomes Structure Each ribosome is formed of two subunits: 1- Small subunit (40s in Eukaryotes, 30s in Prokaryotes): It is binds to the mRNA strand. 2- Large subunit (60s in Eukaryotes, 50s in Prokaryotes): It contains the site of catalysis, the peptidyl transferase centre, which forms peptides bond elongating the polypeptide (rRNA portion). The large subunit has three chambers: 1- A-site, where the amino-acyl tRNA binds. 2- P-site, where the peptidyl transferase activity takes place. 3- E-site, where the tRNA exits the ribosome. Transcription/Translation coupling - In the ribosome, the mRNA is read from the 5’ to the 3’ end. - This directionality is important enabling the coupling (in place and time) of both transcription and translation in prokaryotes. - However, this coupling is not found in the eukaryotes because mRNA is not transported into the cytosol (where the translation occurs) through the nuclear membrane before undergoing post-transcriptional modifications (capping, splicing and polyadenylation). 4|P a ge Translation Translation involves 3 stages: Initiation, elongation and termination. In eukaryotes, the first amino acid to be added in translation is Methionine (AUG). While in prokaryotes it is N-formylmethionine. 1- Initiation of Translation In both prokaryotes and eukaryotes, translation is initiated at a specific site (start codon, typically AUG) located upstream, before, the first codon in the coding region on the mRNA. There are 2 untranslated regions containing non-coding sequences on the mRNA of both prokaryotes and eukaryotes. One is the 5’ terminal upstream from the initiation sites, referred to as 5’ untranslated region (UTR) and the other is downstream at the 3’ terminal, after the stop codon, referred to as 3’ untranslated region. A- In Prokaryotes - Recall that the prokaryotic mRNA is polycistronic; meaning that it encodes for more than one polypeptide within the same mRNA. Before each protein, there is a sequence upstream from the start codon called the Shine-Dalgarno sequence; multiple Shine-Dalgarno sequences are found within a single prokaryotic mRNA. - The 16S rRNA of the small ribosomal subunit has a complementary sequence for this ShineDalgarno sequence. It recognizes it on the mRNA and binds to it signaling the ribosome to scan for the start codon (AUG) to initiate translation. 5|P a ge - After the binding of the small subunit to mRNA, a tRNA molecule carrying an N-formylmethionine binds to the start codon on the mRNA. - This tRNA-mRNA-small subunit (30s) is called the 30S initiation complex. GTP is needed for the formation of this complex in addition to a set of proteins who are initiation factors: IF1, IF2, and IF3. - After the formation of the 30s initiation complex, the large subunit (50s) binds to the complex having the tRNA in the P-site. The complex now is called the 70S initiation complex and translation will start. In summary: Recognition of Shine-Dalgarno sequence → Stable binding between the 30s subunit and mRNA → Formation of the 30s initiation complex → The large subunit binds to the complex → Formation of the 70s initiation complex → Translation begins at the first AUG downstream from the Shine-Dalgarno sequence. B- In Eukaryotes Recall that the eukaryotic mRNA is monocistronic; meaning that the mRNA encodes only one polypeptide for each mRNA molecule. Cap-dependent initiation - There is no Shine-Dalgarno sequence in eukaryotes and another process takes place. Important initiation factors of translation in eukaryotes form a protein complex including eIF4G, eIF4E and poly-A binding protein (PABP). “Refer to the figures” - The eIF4E protein binds to the 7-methylguanosine cap, while the poly-A binding protein (PABP) binds to the poly-A tail. Then, eIF4G links PABP (linked to poly A tail) to eIF4E (bound to the cap) forming a complex at the 5’ cap. - This complex stabilizes the small subunit (40s) on the mRNA which then moves along the mRNA ‘scanning’ for the start codon (AUG). - Once it finds the start codon (AUG), the large subunit (60s) is attached commencing translation elongation. 6|P a ge Cap-independent initiation - In this process, translation does not require the 5’ cap to initiate scanning for the start codon. - The start codon is preceded by a sequence called Internal Ribosome Entry Site (IRES). - The IRES is recognized by either the small subunit (40s) directly, or indirectly by the eIF4G protein followed by recruitment of the small subunit. From the previous points, we can conclude how post-transcriptional modification (capping and polyadenylation) aid in translation: 1- Caps are recognized by the small ribosomal subunit binding to the eIF4E protein initiating translation. 2- Poly-A binds with PABP. Then eIF4G links the poly-A tail to the cap via PABP forming a loop which stabilizes the initiation complex. Note: In the last step of initiation where the large subunit joins the small subunit, this complex will have the P-site occupied with a charged initiating tRNA (typically holding Met) and the A-site will be empty. Re-tracing: The 1st step of translation is initiation. In prokaryotes, the Shine-Dalgarno sequence is recognized by the small subunit (30s) which then moves along the mRNA scanning for the start codon (AUG) where the charged tRNA can bind to in the presence of GTP and initiation factors (IF2 IF3 IF1). Then the large subunit (50s) is added forming the 70s complex commencing polypeptide formation and elongation. In eukaryotes, initiation is either: a- Cap-dependent: Forming a complex of proteins (eIF4G, eIF4E, and PABP) along with the small subunit (40s) at the cap, which then scans for the start codon where then the large subunit is attached commencing elongation. b- Cap-independent: Where an internal ribosome entry site (IRES) is either directly recognized by the small ribosomal subunit or recognized by the eIF4G protein which recruits the small subunit indirectly. 7|P a ge 2- Elongation - During elongation, the amino acids are added to the C-terminus of the polypeptide chain as the ribosome moves from the 5'- 3' end on the mRNA, having the N-terminus as the free end. - Recall, in eukaryotes, the first amino acid to be added is Methionine (AUG). While in prokaryotes it is N-formylmethionine. - The elongation consists of 3 steps that are repeated continuously until the stop codon is reached inducing the termination of the process. These steps are: A- Codon Recognition: Aminoacyl-tRNA, which is charged with a specific amino acid according to the codon recognized, is delivered to the A-site facilitated by an elongation factor (EF-Tu) requiring GTP hydrolysis. The P-site is already occupied with the initiating tRNA. After delivering the charged tRNA to the A-site, the EF-Tu is released to be regenerated again. B- Peptide bond Formation: A peptide bond is formed by peptidyl transferase between the amino acids, having the newly synthesized polypeptide linked to a tRNA in the A-site, leaving the tRNA on the P-site uncharged (inactive). C- Translocation: The ribosome then moves by one codon (3 nucleotides) towards the 3' end translocating the first tRNA (the uncharged) from the P-site to the E-site causing the tRNA to exit the ribosome. The second tRNA (peptidyl-tRNA) is translocated from the A-site to the P-site carrying two amino acid chain, leaving an empty A site. Those steps will be repeated over and over until a stop codon is reached. 8|P a ge 3- Termination The stop codons (UAA, UAG, UGA) are not recognized by any tRNA molecule. Instead, they are recognized by Release Factors. These release factors block the binding of a new aminoacyl-tRNA and facilitate the hydrolysis of the bond between the last amino acid added of the peptide (Cterminus) and the tRNA. Then, the whole complex dissociates. Polyribosomes (polysomes) - A single mRNA molecule can be translated by several ribosomes simultaneously (at the same time) forming a ‘polyribosome’. - This happens in both prokaryotes and eukaryotes. Do not mix this with the transcription/translation coupling process which is present in prokaryotes only. - Each ribosome produces one copy of the polypeptide chain specified by the mRNA. When the protein formation finishes, the ribosome dissociates into subunits that are used in other rounds of protein synthesis. 5’ - Recall that the ribosome reads mRNA in the 5’ - 3’ direction. This means that the ribosome near the 3’ end, having the longest polypeptide formed, is the first ribosome that got attached and started translation from the 5’ end. 3’ 9|P a ge Cloning - Cloning is a molecular biology technique that makes many identical copies of a piece of DNA, such as a gene. - Cloning is used for many purposes such as synthesizing eukaryotic proteins in bacteria, e.g. insulin. By adding the promoter along with the gene of interest into the bacterial plasmid where transcription and translation takes place forming our protein of interest. - Challenges: Insulin is a dimer linked by disulfide bonds and produced from genes containing introns which are not found in bacteria. - Solution: Synthetic DNA is made for each polypeptide, making up the insulin protein, and are inserted into bacteria separately. The bacteria are then cultured, producing many cells. Polypeptides are purified from each bacterial batch and are mixed to form the mature insulin protein. Inhibitors of Translation Some antibiotics work by inhibiting the process of translation in prokaryotes such as: 1- Tetracycline: Blocks binding of aminoacyl-tRNA to A-site of the ribosome. 2- Streptomycin: Induces binding of wrong tRNA-AA complexes causing mRNA misreading resulting in false proteins. 3- Chloramphenicol: Binds to the 50s subunit and inhibits the peptidyl transferase action. 4- Erythromycin: Binds to the 50s subunit and inhibits translocation. In eukaryotes, diphtheria toxin is a protein that interferes with protein synthesis by decreasing the activity of the elongation factor eEF2. 10 | P a g e