Download LS1a Fall 2014 Lab 6: Ribosomal Protein Translation (PyMOL lab #3)

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