BCH401G Lecture 39 Andres

Transcription

1 BCH401G Lecture 39 Andres Lecture Summary: Ribosome: Understand its role in translation and differences between translation in prokaryotes and eukaryotes. Translation: Understand the chemistry of this process, the proteins that are used and their individual roles. Which steps are regulatory? Look at the steps that require energy (GTP or ATP hydrolysis)-why/how is this energy used. Understand how to calculate energy use. What is the relationship between translation rate and fidelity? How would mutations in individual proteins alter the process of translation (think back to our discussion of trna charging)? Overview of Translation: What are key players in translation? We have already talked about two of these: 1. mrna 2. trnas charged with each of the amino acids

2 We need one more thing: 3. The Ribosome. The Ribosome is where the process of translation occurs. Ribosomes are very large complexes composed of rrnas and proteins. In E. coli the large subunit is termed the 50S subunit. The Small subunit is the 30S subunit. 50S subunit and 30S subunit associate to form a 70S ribosome.

3 1. 50S subunit. Complex of two rrnas, 23S rrna and 5S rrna, and 31 different proteins S subunit. Complex of 16S rrna and 21 different proteins. How does the ribosome perform the translation of mrna to protein? 1. Provides a place where codons of mrna can interact with trnas carrying the amino acids designated by the codons. Looks simultaneously at two codons of mrna: 5' GGCGCA ' Gly Ala 2. Then it catalyzes formation of a covalent bond between the amino acids of trnas which are base-pairing to adjacent codons on the mrna. The bond between amino acids is called the "Peptide Bond". A Peptide Bond is formed between the carboxyl group of one amino acid and the Alpha amino group of another amino acid. mrnas have a 5' end and a 3' end - they have Polarity. Proteins also have polarity. 1. The Amino acid at one end of a protein chain has a free alpha amino group. Called "Amino-Terminus" or "N-terminus" of the protein. 2. Amino acid at other end has a free Alpha carboxyl group. Called "Carboxy-Terminus" or "C-terminus" of the protein.

4 Direction of Protein Synthesis is from N-terminus to C-terminus. Each new amino acid is added onto the C-terminal end of growing protein chain. Alpha amino group of the new amino acid (bound to a trna) attacks the carboxyl group of the last amino acid in protein chain. By convention, amino acid sequences are written and numbered left-to-right from N-terminus to C-terminus. Initiation of Translation Have a mrna molecule: 5' ' Ribosome wants to translate the mrna into protein. But there are two problems: Problem 1: What reading frame should be used? In any mrna sequence, there are multiple ways (three reading frames) codons could be read. Each way to read the codons is called a "Reading Frame". Three possible "Reading Frames" Very important for the ribosome to find the correct reading frame. If wrong frame is used -Generate a protein with the wrong amino acid sequence. Not functional.

5 2. Problem 2: At what codon in the mrna does the ribosome begin translation? Turns out both of these problems are solved in the same way. The solution is that the ribosome begins translation at a specific AUG codon within the mrna template termed the "Start Codon". This is a methionine codon, so the first amino acid in proteins is usually methionine. In E. coli the first methionine is modified with a formyl group. Called formyl-methionine (fmet). This special fmet is attached to a unique initiator trna (trnafmet). All subsequent methionines in the protein do not have a formyl group. Only the initiator methionine is modified.

6 But how does the ribosome recognize this particular AUG codon? There are many potential AUG codons in each mrna sequence. Sequences within the 16S rrna, which is part of the small subunit of the ribosome, interact with a complementary sequence in the prokaryotic mrna near the AUG Start Codon. This sequence in the mrna is called the "Shine-Dalgarno Sequence" Interaction of this sequence with the 16S rrna causes the ribosome to begin translation at this particular AUG codon. You can see that starting translation at a specific AUG codon also solves the Reading Frame Problem. Next three nucleotides encode the second codon, and so on.

7 1. The Shine-Dalgarno sequence in the mrna tells the ribosome to initiate translation at a nearby AUG codon. 2. This AUG codon is the first codon of the proper reading frame. Reading frame is now set. This also allows prokaryotes to generate mrnas that encode multiple proteins (operons). Each protein coding region would begin with a S.D. sequence which allows the individual coding regions to be translated. This is not the case for Eukaryotes.

8 Steps of Initiation of Translation. 1. Small subunit (30S) interacts with proteins called Initiation Factors. Initiation Factors help assemble the Initiation Complex. IF-1, IF-2, and IF-3. IF-2 contains a bound GTP nucleotide. Initiator trna (trnafmet) carrying a formyl-methionine joins the initiation complex. Then mrna joins the complex. The 16S rrna of the small subunit identifies the correct AUG Start Codon. The Anticodon of the Initiator trna (fmet) then base-pairs with the AUG Start Codon. This is called the "30S Pre-Initiation Complex".

9 2. The Large subunit (50S) then associates with the Pre-Initiation Complex. Initiation Factors are then released. IF-2 release requires the hydrolysis of the Gamma Phosphate of the bound GTP, converting it to GDP. The phosphate bonds of many nucleotide triphosphates are used to drive the process of translation. Hydrolysis of these bonds liberates energy that can be used to do work. In this case, GTP hydrolysis causes release of IF-2 (drives a change in conformation) from the Initiation Complex. The joining of the 50S subunit with the"30s Pre-Initiation Complex" gives the "70S Initiation Complex". The Ribosome is now ready to begin synthesizing an amino acid sequence from the mrna template. Elongation of Translation. To understand how each amino acid is added to the protein chain, we must look more closely to the region of the ribosome where this process is occurring. Ribosome only looks at two codons of mrna at any one time. Ribosome can only interact with two trnas at any single time. One trna for each codon.

10 The sites where these two trnas bind are called the "peptidyl" site or "P" site and the "aminoacyl" site or "A" site. The Peptidyl site contains the trna that is attached to the growing chain of amino acids. This trna is called the Peptidyl trna. A short chain of amino acids is called a "Peptide". This is why this site is called the "Peptidyl trna" and "Peptidyl" site. The "A" site is where the charged trna carrying the next amino acid binds to ribosome. Three steps in Elongation of Translation: 1. Binding of the appropriate charged trna to the "A" site. 2. Peptide Bond Formation between the amino acid on the trna in the "A" site and the growing amino acid chain attached to the trna bound in the "P" site. 3. Translocation of the Ribosome. The "A" site trna (now carrying amino acid chain) moves to the "P" site. A new"a" site can now be created. The Ribosome moves one codon down the mrna. These processes involve "Elongation Factors" or "EFs". These proteins assist in the elongation steps of translation.

11 Step 1. Binding of charged trna to the "A" site. This involves an elongation factor called EF-Tu. EF-Tu-GTP forms a complex with charged trnas. Like IF-2, EF-Tu also has bound GTP nucleotide. Will write it as EF-Tu-GTP

12 First, the charged trna bound to EF-Tu-GTP enters the "A" site. Another name for a charged trna is an aminoacyl trna. This is why it's binding site on the ribosome is called the aminoacyl site. The ribosome must then examine the different charged trnas that diffuse into the "A" site until it finds one with an anticodon that base-pairs correctly with the codon found in the mrna template. When binding of a proper trna is verified by correct Watson- Crick base-pairing to the codon, EF-Tu releases the charged trna. The timing of this period of analysis is dictated by the rate of GTP hydrolysis and is therefore regulated by EF-Tu (GTPase). The energy for the dissociation of EF-Tu from the charged trna comes from the hydrolysis of the bound GTP to GDP + Pi. So now we write this as EF-Tu-GDP, which is now released from the ribosome complex. A peptide bond can not be formed until EF-Tu is released from the charged trna. It takes time for hydrolysis to occur and for EF-Tu GDP to leave the ribosome. During either period an incorrect charged-trna can be released from the A site, whereas a correct trna will remain bound. There is a balance between the time that EF-Tu remains bound, and its rate of release. A very long binding time would result in very accurate protein synthesis but at a very slow rate, rapid release would result in fast but inaccurate protein synthesis (lots of protein but none of the enzymes would be active due to errors). EF-Tu is then needed to initiate a new cycle of elongation used by binding to another charged trna, but first the GTP-bound form must be regenerated by the action of another Elongation Factor called EF-Ts.

13 EF-Ts causes GDP to be released from EF-Tu and replaced with GTP. This action is termed "guanine nucleotide exchange" or " guanine nucleotide dissociation stimulation" so EF-Ts is a nucleotide exchange factor or dissociation stimulator for EF-Tu.

14 Step 2. Peptide Bond Formation. Once the correct charged trna is present at the "A" site, a peptide bond is formed between the amino acid carried by the trna and the growing amino acid chain attached to the trna bound in the "P" site. Peptide bond formation is catalyzed by the peptidyl transferase activity site of the ribosome (nucleophilic attack of the amino nitrogen on the ester high energy bound formed originally by the aminoacyl-trna synthase of the peptidyl trna). Alpha amino group of the new amino acid attacks the alpha carboxyl group found on the growing peptide chain. As a result, the growing amino acid chain is now attached to the trna found in the "A" site and the chain has been extended by one amino acid.

15 Step 3. Translocation. Translocation involves a third elongation factor called EF-G which also contains a bound GTP. Written as: EF-G-GTP. After Peptide Bond Formation, hydrolysis of the bound GTP to GDP on EF-G causes two things to happen: 1. The "A" site trna (now carrying amino acid chain) moves to the "P" site. The "A" site is now open. 2. At the same time, the Ribosome moves one codon down the mrna molecule. A new charged trna can now enter the open "A" site and interact with the next codon in the mrna. So the GTP hydrolysis on EF-G allows the coordinated movement of the ribosome complex which serves to open a new codon site and allows translation to continue. The Ribosome simply repeats these three steps as each new amino acid is added to the peptide chain. The rate of Protein Synthesis is approximately 18 a.a./second.

16 Termination of Translation Termination involves Proteins called Release Factors: RF-1 RF-2. and RF-3. Elongation of translation occurs until the ribosome reaches a Stop Codon (UAA, UAG, UGA). At this point a Stop Codon is present at the "A" site. The problem is that the cell does not contain a charged trna that is able to interact with these Stop codon sequences. Since there are no trnas that contain the proper anticodon to base-pair with the Stop Codons, extension of the peptide chain is halted. Instead, RF-1 or RF-2 bind to the Stop Codon. RF-1 binds and recognizes either UAA or UAG codon sequences; RF-2 binds to UAA and UGA stop codons. RF-3 interacts with RF-1 or RF-2 and RF-3 also binds GTP: RF-3-GTP

17 Binding of the Release Factors to the Stop Codon leads to the hydrolysis of the amino acid chain from the Peptidyl trna in the "P" site. GTP bound by RF-3 is hydrolyzed to GDP. This causes a conformational change in the RF-3 protein and this causes the Release Factors to dissociate from the Ribosome. Synthesis is complete. The newly synthesized protein leaves the ribosome.

18 Energy Cost of Translation. Translation requires a large amount of energy from the cell. For each amino acid added to the chain, ~4 high energy phosphate bonds are broken. 2N ATP is cleaved to AMP + 2 Pi during charging of the trna with a specific amino acid. Two bonds. 1 GTP for initiation. N-1 GTPs are required for the formation of N-1 peptide bonds. N-1 GTPs are necessary for the N-1 translocation steps. 1 GTP is required for termination. This does not include any proofreading and is therefore a conservative estimate. For a typical 300 amino acid protein: 50,000 kj energy/mole. Translation is a very energy intensive process (single peptide bond +20 kj/mole) because the order of amino acids is critical as is a high degree of fidelity [ different possible ways to put 20 amino acids together to give a 300 aa long peptide]. In some cells, it has been estimated that up to 80% of total available energy (ATP/GTP) is used in protein synthesis. This is why gene expression is so carefully regulated (transcription, translation). Cell loses a lot of energy if it expresses genes it doesn't need.

19 Antibiotics. Antibiotics are used to fight bacterial infections. Many antibiotics inhibit bacterial growth by inhibiting the process of translation. Inhibition specific to translation in bacteria, not eukaryotic cells. So they kill the bacteria infecting you without killing you. Examples: Tetracycline binds to 30S subunit and prevents binding of charged trnas to "A" site. Erythromycin binds to 23S RNA and inhibits the Translocation Step, blocking elongation. The problem with using antibiotics is that microorganisms can develop resistance. This often results from the acquisition of a particular "resistance gene". Erythromycin resistance is gained by the expression of a methylase enzyme that inactivates the drug by blocking its ability to bind to the 23S RNA. Antibiotic resistance is used as a tool in molecular biology (we will see how in the next lecture).

20 Differences between Prokaryotic and Eukaryotic Translation: 1.Eukaryotic Ribosomes are larger and require 4 rrnas. 2.The initiator trna is methionine (not fmet), however as with prokaryotes, the trna is unique and used only to initiate translation. 3.Start signal. No purine rich recognition site, usually the first AUG codon is used. The 5' cap is used to initiate translation and because only one 5' AUG is selected, any single mrna can produce only a single protein. 4. Many more initiation factors are used.