Tutor-marked Assignment 01
Covering : Book 1 Chapters 1−6; Molecular modelling 1–5;
Experimental investigation 1; Reading scientific literature 1.
Cut-off date: Thursday 30 April 2015
Guidance
For advice on capturing pictures from the various activities see the S377
Introduction and Guide, Section 5.1.1. A note on ways to prepare simple
diagrams for eTMAs is available on the Assessment page of the module
website.
This TMA assesses the following module learning outcomes:
Demonstrate knowledge of the molecular biology that underpins cell biology
(Questions 1–5).
Use a sophisticated 3-D molecular modelling program to manipulate and
represent biological molecules in a variety of ways (Question 2).
Design and interpret experiments that use the techniques of SDS−
polyacrylamide gel electrophoresis (SDS−PAGE) and Western blotting
(Question 4).
Critically read primary papers from the scientific literature and be able to
extract and evaluate key information from these papers (Question 5).
The table indicates the types of question in this TMA, and the material each
question covers. It also shows the percentage of the total marks for this
assignment allotted to each question. In the parts of questions where explanations
or descriptions are required, you should aim to give succinct answers of 2 or 3
sentences maximum.
Question Materials covered Marks
1 Chapter 6 10
2 Chapters 2, 3 and Molecular modelling 25
3 Data handling and Chapter 2 10
4 Chapter 3 and Experimental investigation 1 25
5 Chapter 5, Reading scientific literature 30
Question 1
(a) What are the three main classes of membrane lipids? Which of the three is
the most abundant in biological membranes? (2 marks)
(b) Name and explain the key property that membrane lipids have in common
and outline how this property determines the behaviour of membrane lipids in
an aqueous environment. (3 marks)
Copyright © 2015 The Open University WEB 04155 4
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(c) Using stylized representations, sketch the structures that arise when
phospholipids are mixed with water, and explain why building these
structures is energetically favourable. (5 marks)
Question 2
In this question, you will be using Viewerlite to explore the small G-protein Ras
and produce a molecular model. G-proteins are described in Section 3.6.2 and we
recommend that you do this question after you have completed Molecular
modelling 5. At that stage, you should be familiar with all the tools in Viewerlite
that are needed here.
Go to the ‘Library’ folder, within the ‘Molecular models’ section of the DVD,
and open the file 1q21, which shows the catalytic domain of human (H) Ras with
a bound GDP molecule. By examining the structure, answer the following
questions.
(a) What type of secondary structure is adopted by the polypeptide segments
Met 1–Ala 11 and Thr 50–Ala 59? (2 marks)
(b) What type of secondary structure is adopted by the polypeptide segment
Ile 93–Arg 102? (1 mark)
(c) How many chiral centres are present in the C-terminal amino acid residue?
(1 mark)
(d) At the normal cellular pH, what will be the charge of amino acid residue 62?
(1 mark)
(e) In this model of isolated, purified protein none of the cysteine residues has
formed disulphide bonds. Assuming that Ras adopts the same conformation
within the cell, give two reasons why internal disulphide bonds are unlikely
to form in this protein. (2 marks)
(f) The guanine base of the GDP forms hydrogen bonds with Asp 119 in Ras.
(i) How many hydrogen bonds are shown in the model between GDP and
Asp 119? (1 mark)
(ii) What functional group of the amino acid forms the bonds? (1 mark)
(iii) One hydrogen bond is formed between the nitrogen atom N1 in the
guanine base and the oxygen atom OD1 in the Asp residue (N—H—O).
What is the distance between the nitrogen and oxygen atoms? (1 mark)
(g) Is this form of Ras active or inactive? Explain your reasoning. (2 marks)
(h) If site-directed mutagenesis was carried out on H-Ras to change Lys 16 to
Tyr 16, what effect do you think this would have on the function of the
protein? Explain your reasoning. (2 marks)
(i) Outline what effect interaction of Ras with an appropriate GEF (guanine
nucleotide exchange factor) would have on the structure and function of Ras.
(2 marks)
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(j) The segment Ser 127–Tyr 137 adopts an α helix. Draw a molecular model of
this segment of the polypeptide on a white background. The model should
show only amino acid residues Ser 127–Tyr 137. The atoms should be shown
as scaled ball-and-stick, and the C-alpha backbone of the segment using the
‘stick’ representation from the amino-acid style tools. (Omit hydrogen atoms
from the model.) Orientate the model so that the α helix is clearly visible
from the side, and label the two basic amino acid residues, either using the
Viewerlite label function or by adding labels from your word processor, after
inserting the model into your TMA. (9 marks)
Question 3
Ligand A binds to a receptor, R, according to:
A+R ↔ RA
An experiment was carried out to determine the equilibrium dissociation constant
( KD
) for binding of ligand A to receptor R. The following data were obtained.
[RA]/ pmol l
−1
(mg protein)
−1
[A] / pmol l
−1
4.1 0.49
7.8 1.15
15.2 2.14
19.8 3.89
23.5 6.32
28.2 8.67
[RA] is the concentration of ligand bound to the receptor (expressed as ligand
concentration per mg of receptor protein) and [A] is the concentration of unbound
ligand.
(a) Use these data to determine KD
, the equilibrium dissociation constant for the
interaction. (8 marks)
(b) Another ligand, B, also binds to the same receptor, R. However, KD
for the
interaction between B and R is greater than KD
for A and R.
Will the input of free energy required to dissociate RB be greater than or less
than that required to dissociate RA? Briefly set out your reasoning. (2 marks)
Question 4
To answer this question, you will need to use the gel electrophoresis program
(SDS–PAGE) from Experimental investigation 1 on the DVD. Go to the
‘Laboratory’ and from the ‘Options’ menu load the data set for
‘TMA protein 2 (sypherin)’.
The molecular weight markers supplied with sypherin are phosphorylated
proteins with molecular weights: 200 000, 98 000, 68 000, 45 000, 36 000 and
25 000. Note also that the preparation of sypherin is not completely pure.
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Carry out experiments to:
(a) Determine the number of polypeptides in sypherin and their approximate
molecular weight. (It is not necessary to produce a calibration curve to
answer this question, although you may want to do so for practice.)
(5 marks)
(b) Determine whether sypherin is phosphorylated by any of kinases A, B or C.
Your answer to part (b) should consist of a single annotated figure with a
short legend stating what is present in each lane. The answer should also
include a few sentences explaining how you have interpreted the results and
reached your conclusions.
You will need to select appropriate experimental treatments for the samples
and an appropriate gel concentration. You will also need to decide whether a
coomassie-stained gel or a Western blot is needed. Save the gel as a jpeg file
and insert the picture into your eTMA. Annotate the gel/blot to show what is
present in each lane and the markers. (20 marks)
Question 5
You are provided with a copy of the paper to read for this question on the module
website. It can be found in the Assessment page, under Resources.
The full reference for this paper is: Niwa, R. and Slack, F. J. (2007) ‘The
evolution of animal microRNA function’, Current Opinion in Genetics &
Development, 17, pp. 145−150.
This review is concerned with the role of micro-RNAs in evolution. Micro-RNAs
(miRNAs) are a class of short RNA molecule, which regulate protein expression,
primarily by controlling the stabilit y of mRNA. Micro-RNAs are introduced in
Section 5.4.3. They act by binding to regions in the 3ȝ untranslated region
(3 ȝUTR) of mRNAs, which have complementary or near-complementary
sequences. There is some additional useful information ‘Micro-RNA and gene
regulation’ in Book 2 at the end of Section 10.6.4., including Figure 10.44. We
suggest that you tackle this question after you have finished Chapter 5, but skip
ahead to read the relevant subsection in Chapter 10.
All of the information needed to answer this question is in S377 and the text of
the paper. It is not necessary to follow up the extensive reference list.
From your study of S377 and your reading of the review by Niwa and Slack:
(a) Briefly describe five different roles of RNA in cell function. Think of the
different types of RNA and the particular function of each type. (5 marks)
(b) Briefly summarize the structural differences between DNA and RNA.
(3 marks)
(c) How many bases do typical miRNAs consist of ? (1 mark)
(d) What tertiary structure do the precursors of miRNAs have? (1 mark)
(e) What was the first miRNA to be identified? Summarize when during
development it is expressed and what it does. (2 marks)
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(f) The base sequence in mRNA is ‘sense’ and it is produced from the template
strand of DNA. The sequence of a miRNA that binds to a target 3ȝUTR site in
mRNA will be complementary to the mRNA sequence. Suppose let-7
miRNA has the sequence:
5ȝ UGA GGU AGU AGG UUG UAA UAG UU 3ȝ.
What will be the complementary target sequence in the 3ȝUTR of an mRNA
molecule? The sequence should be written 5 ȝ 3ȝ. (2 marks)
(g) What evidence is there that the acquisition of this particular family of
miRNA genes was an important step in the evolutionary development of
metazoans (multicellular animals with differentiated cell types)? (2 marks)
(h) How does the expression of miRNAs vary in different cell types in an
organism? Illustrate your answer with an example from the paper. (2 marks)
(i) Through what mechanisms is it thought that miRNA genes have increased in
higher organisms? What estimate does the paper give for the number of
miRNA genes in the human genome? (It is interesting to relate this to the
number in Section 5.4.3, which was correct as of 2004.) (2 marks)
(j) What is distinctive about the role of miRNAs that are present in vertebrates?
Where is this data shown in the paper? (2 marks)
(k) What evidence do the authors cite, to indicate that miRNAs can affect the
phenotype of adult animals? (3 marks)
(l) Why do the authors think that the 3 ȝ UTR target sequences of some miRNAs
are strongly conserved during evolution, while the majority of miRNA targets
are not conserved? What explanation is proposed for the function of the
genes carrying conserved target sequences? (3 marks)
(m) What is the overall conclusion of the article? (2 marks)