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Download LS1a Fall 2014 Lab 6: Ribosomal Protein Translation (PyMOL lab #3)
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LS1a Fall 2014 Lab 6: Ribosomal Protein Translation (PyMOL lab #3) Goals of the lab: Play around with three-dimensional models of one of biology’s most awesome machines Relate the physical orientation of tRNAs in the A-, P-, and E-sites with the function of each site in the ribosome. Relate the physical orientation of mRNA in the ribosome to the directionality of protein synthesis. Describe how the ribosome makes use of the mRNA triplet nucleotide code. Predict how the binding sites of various antibiotics will affect translation. Required Safety Regulations and Lab Etiquette Wear lab coat and safety glasses at all times Wearing appropriate clothes that protect the leg, foot, and ankle (e.g., jeans, socks, and sneakers) No eating or drinking in lab Introduction As we learned in lecture, the ribosome is the enzyme responsible for translation: the decoding of a sequence of ribonucleotides in a strand of messenger RNA (“mRNA”) into the correct sequence of amino acids of a protein. The ribosome reads the mRNA transcript in the 5’ 3’ direction, and synthesizes an amino acid polymer (a “polypeptide”) in the amino-terminal (“Nterminal”) to carboxy-terminal (“C-terminal”) direction. “Ribosome” is a nifty abbreviation from the enzyme’s initial appellation: the “ribonucleoprotein particles of the microsomal fraction” (try saying that three times fast). It was originally described in this way because just about all that was known about it was that it consisted of RNA (hence the “ribonucleo-”) and protein (hence the, uh, “protein” part of the title). In both bacterial and eukaryotic ribosomes, there is about twice as much RNA than there is protein in terms of mass, and unlike most enzymes the RNA component of the ribosome is primarily responsible for its catalytic activity. We will be looking primarily at the bacterial ribosome today, as it is smaller and slightly less complicated than the eukaryotic ribosome, allowing scientists to study it in far greater structural detail. Both the bacterial and eukaryotic ribosomes consist of a small subunit and a large subunit. The large subunit of the bacterial ribosome is called the “50S” subunit and the small subunit of the bacterial ribosome is called the “30S” subunit. [The names of the subunits are derived from how quickly they migrate to the bottom of a tube during centrifugation. The “S” stands for a “Svedberg,” which is the unit for the rate at which particles of a given size and shape travel to the bottom of a centrifuge tube under as the centrifuge spins. The number in front of the S is the subunit’s “sedimentation coefficient.” Don’t worry, you won’t need to know how these names are derived for the course.] The entire bacterial ribosome, consisting of one small 30S subunit and one large 50S subunit, is called the “70S” ribosome (sedimentation rates do not add up in a linear way) (See Figure 1). Figure 1. The subunit composition of the bacterial (left) and eukaryotic (right) ribosomes. While many core functions of the ribosome are conserved between the bacterial and eukaryotic ribosomes- including peptidyl transfer, codon decoding, translocation, and (we think) the activation of the guanosine triphosphatase (“GTPase”) activity of the elongation factors- the structures of the bacterial and the eukaryotic ribosomes have significant differences. The entire eukaryotic ribosome (the “80S” ribosome) is about 50% larger than the bacterial 70S ribosome. Both the eukaryotic small (“40S”) and large (“60S”) subunits are larger than the bacterial small and large subunits, respectively (See Figure 1). Review of Basic PyMOL Functions Please recall the basic commands that are used to manipulate molecules on the screen using the computer mouse: Rotate (= left mouse button): To rotate the molecule, click on the left mouse button and move the mouse left, right, up or down. Move (= middle button): To “move” an object on the screen, you must click on the object using the wheel as a button. Make sure to hold the button down as you move the mouse and try to not turn the wheel. Turning the wheel simply controls the “Z-axis clipping plane” for the viewer. If you rotate the wheel by accident and clip the image, turn it back in the other direction until the entire molecule reappears. Zoom (= right mouse button): Click on the right mouse button and move the mouse up or down in order to zoom in or out on the molecule. Go easy at first! It doesn’t take much to “lose” the molecule if you go too far. Also, please recall that the rectangular buttons on the right side of the viewer window are used to select “objects” from the list (see example below). Select the object of interest by clicking on the rectangular button to turn it on, or remove it from the screen by clicking on it again to turn it off. We will not be using any of the small square buttons ([A], [S], [H], [L], and [C]) in this lab. Rectangular buttons select objects from the viewing list Procedures for Today’s Lab This lab is composed of three sections; each section will utilize different files. The lab questions (starting in Section 1) are highlighted with a “►” symbol in the left margin of the page and should be answered as you work your way through the different exercises. Please answer your questions on the answer sheet you were given. Your answer sheet will be due a either in section the week of December 3rd – 5th, or in your TF’s dropbox on December 1st. Section 1: Anatomy of the Ribosome Login in as a “Student” using the password “g3n0m3,” with all of the vowels replaced by numbers. You may need to quit LoggerPro when you sign in. Open the “Documents” folder and open the “Life Sciences Labs” folder. Open the “PyMOL-Lab6” folder. (Download and) Open the file “1-Ribosome.pse” [Please exercise some patience: the ribosome is an extraordinarily huge molecule that has pushed the limits of our ability to study its structure. The amount of data in these structural files is therefore huge, so it may slow down the computer at times. If it ever freezes up, please just close the program, re-open the file, and return to the scene that you are interest in.] At the top of your window, click on the “Scene” menu, scroll down, and make sure that “Buttons” is checked: Select scene “001” if it is not already selected. Behold: the ribosome, in all its grandeur! (Neat, eh?) You are now staring at a complete 70S bacterial ribosome that includes a large 50S subunit and a small 30S subunit. The large 50S bacterial subunit consists of a 23S RNA (~2,900 nucleotides), a 5S RNA (~120 nucleotides) and about 30 proteins; the small 30S bacterial subunit consists of 16S RNA (~1,500 nucleotides) and about 20 proteins. As you rotate the ribosome around, you will notice a green part and a cyan part. ► Q1) While it may be hard to tell, the structures we will be looking at today are stunning achievements of science that have taken decades to determine. Many of the structures in today’s lab were cited in the awarding of the 2009 Nobel Prize in Chemistry to three scientists who have produced some of the most remarkable structures of the ribosome. The current view (scene “001”) displays the ribosome using the “sticks” representation, diagramming every atom in the ribosome other than hydrogen. While marvelous, there are so many atoms in the ribosome that it is currently pretty indecipherable what is going on, so we are going to dissect the ribosome apart and then build it back up again. First, we are going to simplify this structure by representing it as a cartoon instead. Select scene “002” The cartoon representation more clearly shows that the ribosome is made of both RNA and ribosomal proteins. The large subunit proteins are cyan, the small subunit proteins are green, and the cartoon representation emphasizes their secondary structure. To more clearly see where the proteins are distributed in the ribosome relative to the RNA, toggle between Scenes 003 and 004. Feel free to rotate and move the ribosome as you do so to get a better spatial feel of how these proteins and nucleic acids are arranged in the ribosome. Scenes 002, 003, and 004 are all in the same orientation (with the large subunit on the left and the small subunit on the right). Select scene “003.” Scene 003 shows only the ribosomal RNAs (“rRNA”). There are only 3 RNA molecules in the entire ribosome: one in the small subunit and two in the large subunit. Select scene “004.” Scene 004 removes all of the ribosomal RNA and instead just shows the ribosomal proteins. The green proteins are those of the small subunit, and the cyan proteins are those of the large subunit. ► Q2) Section 1a: 30S, the small subunit Select scene “005.” This scene hides the large subunit and is now showing the small subunit in what is commonly called the duck view (quack). Can you see why? Figure 2. The “duck” orientation of the bacterial small 30S subunit. In this orientation, we are looking “down” on the small subunit from the “top,” with the large subunit hidden. Now let’s see where the tRNAs bind the small subunit during the process of translation. Select scene “006.” This structure captures a snapshot of the ribosome in the process of translation. If we were to parse translation into three phases: initiation, elongation, and termination; then this is an image of the ribosome in the midst of the elongation phase. There are therefore three tRNAs present in this structure: one in the A-site (magenta), one in the P-site (orange), and one in the E-site (white). The tRNAs are currently shown in the “spheres” representation, which diagrams the van der Waals radius of each atom in a space-filling model. Both the A-site tRNA and the P-site tRNA are charged with an amino acid. The messenger RNA (“mRNA”) transcript that contains the codons to which these tRNAs are bound is cyan and binds along the “neck” of the subunit. Select scene “007.” Scene 007 rotates the small subunit 90 degrees so that we are now starring down the length of the mRNA transcript and the three tRNAs to which the mRNA is bound. Section 1b: The “Decoding Center” of the Small Subunit Select scene “008.” We have now hidden the small subunit so that we can look more closely at the mRNA-tRNA interactions. Note that while the rest of the ribosome is currently hidden, neither the mRNA nor the tRNAs would be interacting in these particular orientations if it were not for the ribosome holding them in the proper places. The tRNAs are now show as sticks, diagramming the location of each of their atoms. Select scene “009,” which rotates our orientation 90 once again. Consider how the tRNAs in the A-site, P-site, and E-site are positioned by the ribosome. Select scene “010,” which focuses our attention on the codon-anticodon pairing occurring the A-site of the ribosome. ► Q3) Select scene “011,” which shifts our focus to the codon-anticodon relationship in P-site. ► Q4) ► Q5) Select scene “012” to shift over to the E-site. The anticodon for the tRNA in the E-site is highlighted in yellow. ► Q6) After examining the “decoding center,” where the ribosome ensures that correct mRNA codon:tRNA anticodon pairing occurs, we will now move to the active site of the ribosome: the peptidyl-transferase center. Section 1c: The Peptidyl-Transferase Center (“PTC”) Once the appropriate amino-acylated tRNA enters the A-site, the ribosome positions the charged amino acid in the A-site to attack the amino-acid in the P-site. The ribosome catalyzes this reaction of adding one amino acid to another by optimizing the proximity and orientation of the amino-acid substrates. Select scene “013.” The 3’ end of every tRNA molecule ends in the sequence “CCA.” When amino-acyl tRNA synthetases attach an amino acid to a tRNA, the amino acid is attached to the 3’ adenosine. The CCA sequence is highlighted in red for the tRNAs in the structure. ► Q7) ► Q8) ► Q9) Select scene “014.” We have now zoomed in on the peptidyl-transferase center (the “PTC”), the active site of the enzyme. The PTC is structurally defined as the place where the amino acid from the A-site is just about touching the amino-acid in the P-site, such that the two reactants are in close enough proximity and in the proper orientation to react and form a bond. The product of the bond will be a poly-peptide bound to the tRNA in the A-site. The reaction is called a “peptidyl transfer” because the peptide chain in the P-site gets transferred to the peptide in the A-site (see Figure 3). Figure 3. The peptidyl-transferase reaction as catalyzed by the ribosome. ► Q10) While we initially approached the PTC by first looking at the small 30S subunit, the PTC actually resides in the large 50S subunit. We will therefore build back up to the entire ribosome to see where protein synthesis takes place and where the newly-synthesized polypeptide exits the ribosome. Select scene “015,” which zooms out and now the mRNA transcript and the tRNAs are now shown as cartoons. The amino acids at the 3’ ends of both the A- and P-site tRNAs are depicted as space-filling models. Select “Scene 016,” which rotates the view 90 degrees. Selecting “Scene 017” makes the small 30S subunit visible again. If you go to the list of objects on the right side of your screen, you will see that “mRNA,” “Asite_tRNA,” “Psite_tRNA,” and “Esite_tRNA” are each highlighted. If you deselect them, you will notice that all of the codon-anticodon interactions we were looking at earlier occur along the groove of the “neck” of the small 30S subunit, the region which we refer to as the “decoding center.” Selecting “Scene 018” returns us to the “duck” view of the small subunit. Select “Scene 019,” which makes the large 50S subunit of the ribosome visible again. Unfortunately as the large subunit is so dense, it obscures where the PTC is. On the right-hand side of your screen, deselect the object “23S_RNA.” As the 23S RNA is the biggest component of the large subunit and it takes up most of the interior space of the large subunit, this allows us to see where the PTC exists within the large subunit. The 23S RNA is primarily responsible for catalyzing the PTC reaction by appropriately positioning the A-site and P-site tRNAs 3’ tails. Notice how far away all of the proteins are from the catalytic center. Select “Scene 020.” This final scene rotates the large subunit into a particular orientation in which you can see the PTC (the spherically-represented amino-acids) through the thistle of the 23S ribosomal RNA. This “tunnel” though the large subunit that connects the PTC to the environment outside the ribosome is called the “polypeptide exit tunnel.” As the protein is synthesized, it is extruded out this channel into either the cytosol or into the ER lumen. The 100-angstrom (Å) long polypeptide exit tunnel emanating PTC is wide enough to accommodate an -helix, but not large enough to allow the formation of any protein tertiary structure. The two proteins highlighted in yellow on the large subunit adjacent to the protein exit tunnel bind a protein chaperone called trigger factor the helps proteins fold as they exit the ribosome. ► Q11) Section 2: EF-Tu and EF-G Close your PyMOL window. Open the file “2-ElongationFactors.pse” We have just examined a structure of the ribosome in the process of elongating a polypeptide strand, but we left out two of the most important players: EF-Tu and EF-G. Section 2a: EF-Tu EF-Tu and EF-G are both GTPases that intrinsically hydrolyze GTP, but they do so slowly unless stimulated by another factor to accelerate their GTPase activity. Select “Scene 01.” We are once again looking at the duck orientation of the small subunit with tRNAs populating the A-, P-, and E-sites. However in this image the A-site tRNA has not fully entered the ribosome and is still bound by EF-Tu (which is dark blue in cartoon representation). The EF-Tu in this image is also bound to GTP. Select “Scene 02.” We are now looking at cartoon representations of EF-Tu (dark blue), which is bound to GTP (shown in sticks) with a Mg2+ ion. Binding to GTP stabilizes EF-Tu in a conformation that has a high affinity for amino acylated-tRNAs. The tRNA is orange and depicted as a cartoon, and the amino acid is shown in the sphere representation at the 3’ end of the tRNA. The mRNA transcript is also shown as a cyan cartoon. Select “Scene 03” to rotate the EF-Tu-tRNA complex. Rotate the structure to see how tightly EF-Tu (in blue) binds the amino acid bound to the tRNA. Select “Scene 04” to zoom in on the GTP molecule and the magnesium ion bound by the beta- and gamma-phosphate groups. Select “Scene 05,” which replaces EF-Tu as stabilized by GTP with the GDP-bound form of EF-Tu. While the shape of EF-Tu when bound to GTP is in blue, the shape of EF-Tu when bound to GDP is in cyan. Select “Scene 06.” This superimposes the blue GTP-bound EF-Tu with the cyan GDP-bound form of EF-Tu. Using the menu on the right, turn off “EFTu_GTP” and turn on “EFTu_GDP.” ► Q12) Now let’s see how EF-Tu interacts with the ribosome as it loads a tRNA into the A-site. Select “Scene 07.” We are once again looking at the small subunit of the ribosome with tRNAs located in the P- and E- sites. However this time the amino-acylated tRNA has yet to enter the A-site. Instead, EF-Tu (colored as a cartoon in blue) is bound to the magenta amino-acylated tRNA and holding it partly outside of the ribosome. This prevents its amino acid from meeting the amino acid bound to the P-site tRNA, thereby preventing its amino acid from getting incorporated into the growing protein until EF-Tu releases the tRNA. Notice how EF-Tu (blue) is holding the 3’ tail of the amino-acylated tRNA (magenta) outside of ribosome. Select “Scene 08” to zoom in on the A-site codon-anticodon interaction. ► Q13) Select “Scene 09” to return to the structure of EF-Tu prior to loading a tRNA into the A-site. Select “Scene 10.” Scene 10 replaces the structure of EF-Tu loading an aa-tRNA with a structure of EF-G in the process of shifting the tRNAs in the A- and P-sites into the P- and Esites, respectively. This step happens after the peptidyl transferase reaction occurs, and opens up the A-site so that the next amino-acylated tRNA can enter. The small subunit is now colored white, the P-site tRNA is colored yellow, and the E-site tRNA is colored pale green. EF-G is shown as a cartoon in red. How does the structure of EF-G interacting with the ribosome compare with that of EF-Tu-bound to a aa-tRNA interacting with the ribosome? Select “Scene 11” to overlay the structure of EF-Tu bound to a tRNA with the structure of EFG. Scene 11 structurally aligns the EF-Tu-(aa-tRNA) complex and the EF-G complex. The two are very structurally similar, which allows them to both interact with similar components of the ribosome as they drive the steps of the elongation cycle. Comment to your lab partner about how amazing and beautiful it is that EF-Tu bound to an amino-acylated tRNA has the same shape as EF-G, and that both factors can therefore interact with the ribosome in very similar ways. Using the menu on the right, turn “EFTu_tRNA” on and off, and “EFG” on and off. How do the two structures relate? Section 3: Antibiotics While the bacterial and eukaryotic ribosomes both perform many of the same functions, they have significant structural differences. The entire eukaryotic 80S ribosome is about 4.3 Megadaltons (1 x 106 Daltons, and a carbon atom is 12 Daltons) 50% larger than the bacterial 70S ribosome. Both the eukaryotic small (“40S”) and large (“60S”) subunits are larger than the bacterial small (“30S”) and large subunits (“50S”), respectively. Moreover, only about one-third of the 80-90 ribosomal proteins have bacterial counterparts. Because the bacterial ribosome is so structurally different than the eukaryotic ribosome, drugs targeted to inhibit the bacterial ribosome have only limited cross-reactivity with our eukaryotic ribosomes. We can therefore take antibiotics that inhibit bacterial ribosomes that leave our ribosomes unaffected, greatly reducing the threat of side effects. As ribosomes are critical for life (inhibiting protein production kills the cell), antibiotics which target the ribosome can both effectively kill bacterial cells and limit the damage incurred by our eukaryotic cells. In fact, about 50% of pharmaceutically useful antibiotics target the ribosome. (Download and) Open the file “3-Antibiotics.pse” Our first image is once again that of the duck view of the small 30S subunit. Select “Scene 002.” Scene 002 indicates where the antibiotic tetracycline binds. ► Q14) Select “Scene 003.” We are now looking at the duck orientation of a small subunit again, but this time with tRNAs bound to the A-, P-, and E-sites. Select “Scene 004” to show the large subunit proteins and the 5S RNA. The major component of the large subunit, the 23S RNA, is still hidden. Select “Scene 005” to show the binding site of the antibiotic erythromycin (it is displayed as space-filling spheres and is colored green). Select “Scene 006” to show the binding site of the antibiotic chloramphenicol (it is displayed as space-filling spheres and is colored red). ► Q15) Codon Table