C It
Resume:
Due: Thursday, Oct. 24 before class
Before attempting the problems, first read the following recent review paper from one of the Nobel
prize winners: T. M. Schmeing and V. Ramakrishnan. What recent ribosome structures have
revealed about the mechanism of translation. (2009) Nature 461:1234–1242.
Problem 1: Ribosome structure and function
For this problem, you will need to load PDBs 2WDK.pdb and 2WDL.pdb in VMD. Note that the
PDBs each contain distinct parts of the ribosome, one with the large subunit, and one with the
small subunit and other associated molecules.
(a) What are the three primary steps in translation? Describe each in 1-2 sentences.
(b) Identify the 3 tRNAs in the structure. Also, identify the amino acid the P-site tRNA is charged
with. Make a figure highlighting these (the 3 tRNAs and P-site amino acid) and label them. (Hint:
the PDB header includes a list of segments and their corresponding chain names.)
(c) The provided file “exittunnel.tcl” contains an atom selection macro for those residues surround-
ing the polypeptide exit tunnel. Source this file (“source exittunnel.tcl”) in the Tk console of
VMD. Now using the keyword “exit tunnel”, make a graphical representation of the exit tunnel.
Also make a representation for the P-site tRNA, which is positioned at the entrance to the tunnel.
Make a figure of the large subunit, highlighting the exit tunnel, and the P-site tRNA.
(d) The ribosome has been proposed to function much like a Brownian ratchet (see for example the
paper Cornish, P. V., Ermolenko, D. N., Noller, H. F., and Ha, T. (2008) Spontaneous intersubunit
rotation in single ribosomes. Mol. Cell 30:578–588). What is the function of ratcheting in the
ribosome? Why does a true Brownian ratchet violate the 2nd law of thermodynamics? How does
the ribosome address this limitation? (Hint: consider what acts as the pawl.)
Problem 2: Initiation
In this problem, you will examine mRNA and the initiation process. You will need to load the
PDBs 2HGP.pdb and 2HGQ.pdb in VMD.
(a) The Shine-Dalgarno sequence is an mRNA sequence upstream of the coding region that is
recognized directly by the ribosome, ensuring proper positioning of the mRNA prior to translation.
The consensus sequence in bacteria is AGGAGG. Find this sequence in the structure and the
corresponding anti-Shine-Dalgarno sequence in the 16S rRNA. What is the anti-Shine-Dalgarno
sequence and what are its residue numbers in this structure? (Hint: You may again find the PDB
header useful for identifying chain names. The sequence viewer in the VMD Extensions Analysis
menu may also help.)
(b) The following mRNA sequence codes for a seven residue protein. Note that it’s not uncommon
for mRNA to possess long 5’ and 3’ untranslated regions (UTRs, even hundreds to thousands of
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bases in length), which often serve additional regulatory roles.
1 GGAUGCGGGG AAAAAAGGGC UCCUUUUGGG GGGUUUUCCC CGCACCGGGC
51 GGACCUGGGC GGAGAGGAAA CGCGGCAACU CGCCCGUCUC GGGUUCCCGC
101 CCACGACCCU UAAGGAGGUG UGAGGCAUAU GGAAUCGAGU GCUGGGGAGU
151 AACCCCUGAC GGUCCCGCCC CCGCCCCCUU UGGGGGGGCG GGGGGUUUUU
What is the start position of the Shine-Dalgarno sequence? The start and stop codons? What is
the protein sequence produced by this mRNA? (Hint: It spells out a word.)
(c) Assume the base at position 142 gets mutated from C to G. What is the result for the protein?
Will this mutation likely have a positive, negative, or neutral effect for the protein’s function?
Why?
(d) Now assume the base at position 132 gets mutated from G to A. What is the result for the
protein? Will this mutation likely have a positive, negative, or neutral effect for the protein’s
function? Why?
(e) Colicin E3 is a toxic bacteriocin that functions by cleaving the 16S rRNA between A1493 and
G1494. How does this kill the bacterial cell?
Problem 3: Decoding
For this problem, you may continue using PDBs 2HGP.pdb and 2HGQ.pdb.
(a) While the genetic code has some redundancy built in, the ribosome still needs to be able to
discriminate between some tRNAs differing by only one base in the anticodon when matching them
to the mRNA codon (cognate vs. near-cognate). A mismatch of one base pair (for example, CUU
vs. UUU) results in the loss of a single hydrogen bond; let us assume the resulting free energy
difference is 2.5 kcal/mol. What is the minimum error rate possible for this free energy difference?
Is codon-anticodon recognition sufficient to explain a typically measured error rate of 5 10 3 ?
(b) A near-cognate instead of cognate tRNA results in the loss of an additional hydrogen bond
between the aforementioned three 16S rRNA bases and the A-minor groove of the codon-anticodon
helix. Including this interaction, what is the error rate now? How does this compare to the
measured error rate? What are possible reasons for the discrepancy? (At least one is given in the
paper provided.)
(c) In order to further enhance fidelity, the ribosome carries out a proofreading step after initial
tRNA selection and GTP hydrolysis by EF-Tu. In Ref. 27 of the provided paper (Blanchard, S.
C., Gonzalez, R. L., Kim, H. D., Chu, S. and Puglisi, J. D. (2004) tRNA selection and kinetic
proofreading in translation. Nature Struct. Mol. Biol. 11:1008-1014), the authors use FRET to
separate the different stages of tRNA selection. FRET efficiencies of 0.35, 0.5, and 0.75 are taken
to represent initial sampling of the tRNA, initial selection resulting in GTP hydrolysis, and full
accommodation, respectively. They found the following data:
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cognate near-cognate
number observed at FRET = 0.35 150 755
number that reach FRET 0.50 97 81
number that reach FRET = 0.75 84 3
The error rate for a given step is the ratio of the rates of near-cognate and cognate tRNA selec-
tion; for example, the overall error rate found in this study is 7.1 10 3 . Determine the individual
error rates for initial tRNA selection and the subsequent proofreading step. The large difference
( four-fold) in error rates is a result of the long time required for full tRNA accommodation after
GTP hydrolysis.
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