ncbs comprehension exam, 2013 june answers

7/25/2019 NCBS Comprehension Exam, 2013 June Answers

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1. Errors in replication will effect the survival of the organism maximally. There may

be only one or two copies of a gene and this is utilized to generate a number of mRNAmolecules. Each of these mRNA molecules is utilized multiple times to generate the

corresponding polypeptide molecules. Since there is amplification in the number of

molecules that carry information from the gene, errors during transcription andtranslation will generally get diluted out. However errors during replication can get fixed

as mutations and have an adverse effect on the corresponding polypeptide or theregulation of genes located in the linear and spatial vicinity. (3 marks)

2. Strategies to ensure translational accuracy:a. Accurate coupling of amino acids to correct tRNA molecules by aminoacyl tRNA(Aa-tRNA) synthetases. These enzymes read the anticodon on tRNA exploit the

chemical nature of the side chain of an amino acid to ensure that the correct amino acidis coupled to the corresponding tRNA. In the case of isosteric side chains, a double

sieve mechanism is utilized to prevent the covalent coupling of the wrong amino acid.

b. Codon-Anticodon recognition, all three bases have to pair to ensure maximumfidelity. The interaction of the codon and anticodon is probed by RNA bases of specificnucleotides within the ribosome. The interactions between nucleotides of the ribosome

and the minor groove atoms of the base pair formed from codon-anticodon pairing will

only appear if Watson-Crick base pairs are formed between the bases of the codon andanticodon.

EF-Tu undergoes GTP hydrolysis and releases tRNA in the A-site only whenall three bases are paired. Any mismatch in the codon-anticodon pairing will effect thepositioning of EF-Tu and it will be unable to access the factor binding centre where GTP

hydrolysis occurs.

Once Ef-Tu is released, tRNA undergoes a conformational change to attain aposition that will allow the peptidyl coupling to occur (this is termed accommodation). Ifthe codon-anticodon interaction is suboptimal, this reorientation will result in release of

the incorrect tRNA. (4 marks)

3. Experiments to test these strategies would involve making site directed mutations inthe (i) anticodon recognition site of Aa tRNA synthetases (ii) codons and anticodons of

mRNA and tRNA, respectively (iii) in the nucleotides of the ribosome involved inmonitoring the formation of Watson-Crick base pairs on codon-anticodon pairing. The

level of incorporation of the wrong/right amino acid in the presence of wt and mutantmRNA/rRNA/AatRNA-synthetase can then be monitored. Additionally, binding

experiments can be done with labeled wild type and mutant Ef-Tu to monitor changes in

the conformation of this translation factor. Also, labeled tRNA can be utilized to monitorchanges in the conformation of tRNA during various steps of the coupling process. (4

marks)


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Comprehensive exam Part A:

1. [Calculator required] An antibody-based diagnostic kit can operate in three

different modes, shown to have the following false-positive (FP) and false-negative

(FN) rates:

Mode FP FN

A 0.005 0.5

B 0.15 0.15C 0.01 0.5

You plan to use these kits to screen for infections in a certain population. You already

know that Infection-1 has an incidence of 5% and Infection-II has an incidence of

25%, and that the two are not correlated. You can only test each individual once for

each infection.

Which mode of testing would be most informative when testing for: Infection-I?Infection-II?

This is a quantitative question, show all your calculations. The table of logarithms in

base 2 might be useful.

log2(x)

x 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09

0 inf. -6.64 -5.64 -5.06 -4.64 -4.32 -4.06 -3.84 -3.64 -3.47

0.1 -3.32 -3.18 -3.06 -2.94 -2.84 -2.74 -2.64 -2.56 -2.47 -2.4

0.2 -2.32 -2.25 -2.18 -2.12 -2.06 -2 -1.94 -1.89 -1.84 -1.79

0.3 -1.74 -1.69 -1.64 -1.6 -1.56 -1.51 -1.47 -1.43 -1.4 -1.36

0.4 -1.32 -1.29 -1.25 -1.22 -1.18 -1.15 -1.12 -1.09 -1.06 -1.03

0.5 -1 -0.97 -0.94 -0.92 -0.89 -0.86 -0.84 -0.81 -0.79 -0.76

0.6 -0.74 -0.71 -0.69 -0.67 -0.64 -0.62 -0.6 -0.58 -0.56 -0.54

0.7 -0.51 -0.49 -0.47 -0.45 -0.43 -0.42 -0.4 -0.38 -0.36 -0.34

0.8-0.32 -0.3 -0.29 -0.27 -0.25 -0.23 -0.22 -0.2 -0.18 -0.17

0.9 -0.15 -0.14 -0.12 -0.1 -0.09 -0.07 -0.06 -0.04 -0.03 -0.01

Ans:

Clearly you would never use Mode C since Mode A is better in every case. The

choice is between Modes A and B. Supposeprepresents the infection incidence, qis

the FP rate, ris the FN rate. We then have the following table:

Not infected InfectedTests negative (1-q)(1-p) rp


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Tests positive q(1-p) (1-r)p

The correct test to use would be the one that maximizes mutual information. If X

represents the state of infection and Ythe results of the test, then we want to calculate

I(X;Y) =H(X) +H(Y) H(X,Y)

The result of this calculation (in bits) is:

Mode A Mode B

Infection-I 0.10 0.27Infection-II 0.08 0.30

So you would use Mode A to test for Infection-I and Mode B to test for Infection-II.


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3. If we allo ssRNA with loops into the answer, then we might say that RBP101 binds

to a particular RNA secondary (loop) structure. Since many sequences could give the

same secondary structure, this would produce no consensus.

Now, experiments to test. There are several options. I will only discuss those forhypothesis (3) above since that is the one most people used in their answers. There are

3 important parts.

a. First verify that all known binders do have the same loop structure.

b. Mutate a known binder to change its loop structure and show that binding no longeroccurs.

c. Construct an artificial sequence with the same loop structure and show that it still

binds.


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3. There are many possible definitions of a protein domain. Give a reasonable

scientific definition in each of the following categories, and also a test (algorithm or

experiment) to determine the domain breakup of the protein according to each

definition:

a. Structural b. Functional c. Folding d.

Evolutionary

Ans:

The whole point of this question is to realize that we usually use the term domain

very loosely, relating to many overlapping but not equivalent definitions. This loose

usage leads to sentences such as the DNA-binding fold is a highly conserved

domain.

a. This definition pertains only to the proteins structure. Look at the contact map, seeif any parts of the protein form interacting blocks of residues. Or just look at the

protein structure and see if you can visually discern large independent blocks. Breakthe protein up according to these blocks.

b. This definition pertains to a combination of biochemistry and genetics. Set some

assay for function. Now break off chunks of the protein and see if the function is

preserved. As we move between different functions we can hopefully ascribe function

to the majority of the protein. This is the most problematic definition.

c. This definition pertains to the dynamics of folding. During the folding process,

check (by experiement or simulation) if large numbers of residues make contact in a

cooperative manner. These would be domains by the folding definition, though theymay be smaller or larger than the traditional structural domains. Alternatively, you

could define it as the minimal stretch that is able to fold independently of the rest ofthe protein.

d. This definition pertains only to global sequence conservation. Look at all proteins

in the universe. Find conserved bits between all protein pairs. This will tend to

produce smaller and smaller breakups but the process will end at some point where

the conserved bits get no smaller. This is the minimal irreducible conserved part of

proteins. This is the evolutionary domain. E.g. this is the definition pfam uses.


6/16

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10/16

Q7 Model answer

The following points should be covered.

1. Application of H should result in a change in the concentration of X that is measured in vitro or in vivo.

2. Applying X in the absence of H should result in an increase in cell size. (may indicate independent

roles in increasing cell size)

3. Preventing the production of X or inhibiting its function should block the effect of applying H with

respect to increase in cell size. (suggest that X is required for the effects of H on cell size)

Describing various technologies without clearly articulating the above points is insufficient.

Q8 model answer

Answer must have elements of each of the following mechanisms.

1. E1 is localized in compartment B and E2 is excluded from it.

2. E2 is present in all other compartments other than B and E1 id excluded from them.

3. E1 and E2 are present in all compartments but E1 is optimally active in B and E2 is inactive/inhibited

in B. mechanisms for this to be included.

4. Transporters enrich S in compartment B by actively enriching it in this compartment or there are

mechanisms that prevent S from leaving compartment B.


11/16

Q. Examine the following two assertions and design an experiment, in each case, that provides proof for the

assertion:

a. A regenerating salamander limb always does so in the distal direction and there is no attempt to replace

missing parts.

b. The Hydra head-region produces a substance inhibitory for regeneration that falls off with distance from

the head.

Your answers should be written out and please NOT use figures!

Answers: To simple and elegant experimental designs are illustrated below. They need to write out the

answer


12/16


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ncbs comprehension exam, 2013 june answers

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