Some Issues in Molecular Cell Biology - Assignment Example

Question a)What are the three main es of membrane lipids? Which of the three is the most abundant in biological membranes? (2 marks) The three major kinds of membrane lipids are phospho-lipids, glycolipids, and cholesterol. Moreover, Phospholipids are the most abundant in all biological membranes. These have a polar head group and two hydrophobic hydrocarbon tails. The tails of phospholipids are usually fatty acids, and they can differ in length (they normally contain between 14 and 24 carbon atoms). By definition, lipids are water-insoluble biomolecules that are highly soluble in organic solvents such as chloroform. Lipids have a variety of biological roles: they serve as fuel molecules, highly concentrated energy stores, signal molecules, and components of membranes. (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) Membrane lipids possess a critical common structural theme: membrane lipids are amphipathic molecules (amphiphilic molecules), most of which form bilayers. Membrane lipid contains both a hydrophilic (water loving) and a hydrophobic moiety. In essence, hydrophilic molecules dissolve readily in water because they contain charged groups or uncharged polar groups that can form either favorable electrostatic interactions or hydrogen bonds with water molecules. Hydrophobic molecules, by contrast, are insoluble in water because all, or almost all, of their atoms are uncharged and nonpolar and therefore cannot form energetically favorable interactions with water molecules. Question 2 (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) (You need to upload the relevant data). Question 3 (a)Use these data to determine KD, the equilibrium dissociation constant for the interaction. (8 marks) [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.Therefore, KD = [A]pmol l−1 / [RA] pmol l−1 (mg protein) −1 [RA]/pmol l−1 (mg protein)−1 [A]/pmol l−1 KD 4.1 0.49 0.1195121951219512 7.8 1.15 0.1474358974358974 15.2 2.14 0.1407894736842105 19.8 3.89 0.1964646464646465 23.5 6.32 0.268936170212766 28.2 8.67 0.3074468085106383 (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) In my opinion, the free energy required to dissociate RB will be greater since it follows that the stronger the bond formed between the ligand (B) and receptor (R) the more the free energy is required to dissociate RB. In addition, KD for the interaction between B and R is greater. Question 4 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) (You need to upload the relevant data). Question 5 (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) RNA molecules are single strands containing the nucleotides: adenine (A), guanine (G), cytosine (C), and uracil (U). Often, RNA molecules form secondary structures much like proteins and may interact with DNA, other RNA molecules and proteins. These interactions help define the particular function of each type of RNA, as tabulated below. Type of RNA Function Messenger RNA (mRNA) Tells ribosomes the order in which to add amino acids to the growing protein chain Ribosomal RNA (rRNA) Part of the ribosome that “reads” mRNA Transfer RNA (tRNA) Brings amino acids to the ribosome and helps the ribosome insert the amino acid into the growing protein chain Heterogeneous nuclear RNA (hnRNA) Unprocessed or partially processed transcripts not yet ready for use. Small nucleolar RNA (snRNA) 60-300 bases. Help with chemical modification of rRNA, tRNA, and snRNAs. (b) Briefly summarize the structural differences between DNA and RNA. (3 marks) Although RNA is chemically very similar to DNA, the three dimensional structures are very diverse. Whereas the DNA almost exclusively displays the classical Watson-Crick double helix structure, RNA exhibits a wide-range of structural folds. Some RNA molecules have a defined, unique conformation that is crucial to their function, while other molecules may have a more flexible conformation. Chemically, the difference between DNA and RNA appears to be negligible. Whereas DNA is constructed from deoxyribonucleotides, RNA consists of ribonucleotides, the difference being a single oxygen atom attached to the 2’ carbon of ribose sugar ring. Another difference is in the selection of bases: RNA makes use of uracil instead of thymine, which are identical except for the methyl group in thymine.In addition, another important difference is that RNA does not have a complementary strand and is most often single stranded. However, RNA often contains stretches of self-complementary sequences that enable it to fold back on itself, forming stretches of hair-pin loops that can adopt a double helical structure. (c) How many bases do typical miRNAs consist of ? (1 mark) MicroRNAs (miRNAs) are small non-coding RNAs (ncRNAs) with approximately 20 nucleotides (nt) length that regulate gene expression posttranscriptionally by binding to 3’untranslated regions (UTR), coding sequences or 5´UTR of target messenger RNAs (mRNAs) and leading to inhibition of translation or mRNA degradation. It is predicted that miRNAs regulate approximately 30% of the human protein-coding genome. miRNAs control the expression of genes involved in several biologic processes such as apoptosis, proliferation, differentiation and metastasis. (d) What tertiary structure do the precursors of miRNAs have? (1 mark) Most precursors of miRNA (pre-miRNA) have a hairpin stem tertiary structure. Both plant and animal miRNAs are cleaved from one arm of foldback precursors (pre-miRNAs). It is generally accepted that pre-miRNA secondary and/or tertiary structures are critical in miRNA biogenesis. (e) What was the first miRNA to be identified? Summarize when during development it is expressed and what it does. (2 marks) Lin-4 was the first miRNA to be discovered, in 1993, by the joint efforts of Victor Ambros’s and Gary Ruvkun’s laboratories. In the nematode C. elegans, heterochronic genes control temporal development pattern of all larval stages. One of these genes is lin-4, discovered by the isolation of a null mutation that causes a failure in temporal development. Animals with lin-4 loss-of-function mutations are missing some adult structures, are unable of laying eggs and reiterate early development programs at inappropriate late larval stages. Lin-4 activity is required for the transition from L1 to L2 stage of larval development. However, since there is no homolog to lin-4 in other species, this discovery was considered to be unique.  Bibliography Almeida, M. I., Reis, R. M., & Calin, G. A. (2011). MicroRNA history: discovery, recent applications, and next frontiers. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 717(1), 1-8. Berg, J. M., Tymoczko, J. L., & Stryer, L. (2002). There Are Three Common Types of Membrane Lipids. JANSON, L. W., & TISCHLER, M. E. (2012). The big picture medical biochemistry. New York, McGraw-Hill. http://www.accessmedicine.com/resourceTOC.aspx?resourceID=761. Krol, J., Sobczak, K., Wilczynska, U., Drath, M., Jasinska, A., Kaczynska, D., & Krzyzosiak, W. J. (2004). Structural features of microRNA (miRNA) precursors and their relevance to miRNA biogenesis and small interfering RNA/short hairpin RNA design. Journal of Biological Chemistry, 279(40), 42230-42239. LILJAS, A. (2009). Textbook of structural biology. Hackensack, NJ, World Scientific Ni, M., Shu, W., Bo, X., Wang, S., & Li, S. (2010). Correlation between sequence conservation and structural thermodynamics of microRNA precursors from human, mouse, and chicken genomes. BMC evolutionary biology, 10(1), 329. Niwa, R., & Slack, F. J. (2007). The evolution of animal microRNA function.Current opinion in genetics & development, 17(2), 145-150. MacFarlane, L. A., & Murphy, P. R. (2010). MicroRNA: biogenesis, function and role in cancer. Current genomics, 11(7), 537. RICHARDS, J. E., & HAWLEY, R. S. (2010). THE HUMAN GENOME a Users Guide. Burlington, Elsevier Science.http://public.eblib.com/choice/publicfullrecord.aspx?p=647537. http://www.sabiosciences.com/pathwaymagazine/pathways7/microrna.php Read More