[I] MCB 3201 Gene Expression

1. [I] MCB 3201 Gene Expression • Instructor: Dr. Thomas T. Chen • Office: TLS Rm 413A; Te: 860-486-5481; E-mail: Thomas.Chen@uconn.edu; Office hour: Tue 11:00 a.m. - 1:00 p.m. or by appointment • Class Meeting Time: Tue and Thu 9:30 – 10:45 a.m. in TLS 263 • Text Book: • Lewin’s Genes X (by Krebs, Goldstein and Kilpatrick) • Course Website: www.sp.uconn.edu/~ttc02001/MCB3201/ • A recommended extra-reading: • RNA, Life’s indispensable molecule (by James Darnell), pubnlished by Cold Spring Harbor Laboratory Press (can be purchased through amazon.com) • Course Grade: • Average of two in-class exams

2. MCB 201 Gene Expression (II) • Two in-class Exams: • Exam I: Thu, 03/04 (Tue) • Exam II: Tue, 04/29 (Tue) • I will lecture 75 minutes in each lecture slot • Exam questions will consist of definitions, short and long answers and problem solving questions. Exam materials will be taken from lecture slides, assigned pages in the textbook and assigned papers on the MCB 3201 website • The course grade will be determined by averaging the scores of two exams

3. Some Facts About Gene Expression in Eukayotes • The central dogma of molecular biology is that DNA produces RNA through transcription which in tern produces proteins through translation • Different tissues and cell types of the same organism show differences in the different proteins which are present and in their relative abundance • Similarly, different tissues and cell types show differences in the mRNAs which are present and in their relative abundance • Unlike the contents of mRNA and proteins, the content of DNA of different tissues and cell types is the same in a specific species of organism • Therefore, gene control must operate to produce different mRNA population in different cell types from the same DNA through regulation at: • Transcriptional level • Post-transcriptional level • Translational level

4. [I] Principle of Supramacromolar Assembly in the Biological System An important principle in the biological system

5. Chemical Composition of Living Cells • Hydrogen, oxygen, nitrogen, carbon, sulfur, and phosphorus normally makeup more than 99% of the mass of living cells • About 70% percent by mass of the molecules inside living cells are water molecules • Cells normally contain more proteins than nucleic acids (DNA & RNA) • Cells also contain carbohydrates, saturated and unsaturated fatty acids, steroids, cholesterol, lipids, amino acids and inorganic elements • An important question: How are these compounds associate together to form cells with specific structures and functions? How is regulation of gene expression achieved?

6. Types of Biochemical Bondings • Covalent bonding: -50 to -100 Kcal/mol • Ionic bonding: -1 to -80 Kcal/mol • Hydrogen bonding: -3 to -6 Kcal/mol • Van der Wallas attraction: -0.5 to -1 Kcal/mol • Hydrophobic interaction: -0.5 to -3 Kcal/mol • Weak chemical interactions: ionic bonding, hydrogen bonding, Van der Walls interaction and hydrophobic interaction

7. Amino Acids • Different protein molecules are made up of the same 20 natural occurring amino acids but with specific sequence • Each amino acid contains two functional groups: amino group and carboxyl group

8. Unique Property of Amino Acids • The pH of an amino acid solution at Zwitterion form is called isoelectric point of the amino acid • Why amino acids or proteins can serve as a good buffer? Zwitterion Isoelectric point

9. Nonpolar Amino Acids Nonpolar amino acids contain R groups that are non-polar in nature. Of 20 amino acids, 9 amino acids are non-polar

10. Polar or Charged Amino Acids

11. Types of Chemical Bonds in Biologically Important Molecules (I) • Covalent bond: bond strength -50 to -100 Kcal/mol

12. Types of Chemical Bonds in Biologically Important Molecules (II) • Ionic bond: bond strength -1 to -80 Kcal/mol Bonds formed between the charged amino group of basic amino acids (lys, arg, and his) and the charged carboxyl group of acidic amino acids (asp and glu)

13. Types of Chemical Bonds in Biologically Important Molecules (III) • Hydrogen bond: -3 to -6 Kcal/mol

14. Types of Chemical Bonds in Biologically Important Molecules (IV) • Van der Waals attraction: -0.5 to -1 Kcal/mol The electron cloud around any nonpolar atom will fluctuate, producing a flicking dipole. Such dipoles will transiently induce an oppositely polarized flickering dipole in a near-by atom. This interaction generates an attraction between atoms that is very weak. However, since many atoms can be simultaneously in contact when two surfaces fit closely, the net result is often significant

15. Types of Chemical Bonds in Biologically Important Molecules (V) • Hydrophobic interaction: -0.5 to -3 Kcal/mol Nonpolar amino acids: gly. Leu. Ilu, val, ala, trp, met, phe, pro

16. Levels of Structures of Proteins • Primary structure: Peptide bond formation (covalent bonds) • Secondary structure: Hydrogen bonding form within one polypeptide chain(a-helical and b-sheet structure) • Tertiary structure: Ionic interaction, hydrophobic interaction, hydrogen bonding and Van der Waals attraction formed among moieties within one polypeptide chain • Quaternary structure: Weak chemical interactions among different polypeptide chains • Supramolecular assembly of macromolecules: Weak chemical interactions of different macromolecules

17. Making a Peptide Chain • When the carboxyl group of one amino acid is brought adjacent to amino group of another amino acid, an enzyme (peptide synthetase) can catalyze an dehydration reaction to form a peptide bond • When this reaction is repeated over and over, a polypeptide will be formed

18. The a-Helical Structure of a Polypeptide Hydrogen bond is formed between the N-H of every peptide bond and the C=O of a neighboring peptide bond located four peptide bonds away in the same chain

19. The b-Sheet Structure of a Polypeptide Individual peptide chains run in opposite directions and hydrogen bonds are formed between peptide bonds in different strands Structure of a b-turn

20. Tertiary Structure of a Polypeptide • Chemical properties of the side chains of amino acids help define the tertiary structure of a peptide • Disulfide bonds between the side chains of cysteine residues in some proteins covalently link regions of proteins, thus help to stablize the tertiary structure of a protein • Amino acid with charged hydrophilic polar side chains tend to in the outside surface of proteins, by interacting with water molecules, can help proteins to be soluble in aqueous solutions and form non-covalent interactions with other water-soluble molecules • Amino acids with hydrophobic nonpolar side chains are usually sequestered away from the water-facing surfaces of a protein, forming a water-insoluble central core • Proteins usually fall into one of the three broad categories based on their tertiary structure: fibrous proteins, globular proteins and integral membrane proteins

21. Tertiary Structure of a Polypeptide • Tertiary structure refers to the overall conformation of a polypeptide chain – that is the three dimensional arrangement of all its amino acid residues • Tertiary structure is stabilized by hydrophobic interaction between non-polar side chains, and hydrogen bonding of polar side chains and peptide bonds • Since the stabilizing interactions are weak, the tertiary structure of a protein is not rigidly fixed, but undergoes continual, minute fluctuations

22. Motifs of Protein Secondary Structure • Structural motifs are regular combinations of secondary and tertiary structures of proteins • Any particular structural motif often performs a common function in different proteins • The primary sequences responsible for a given structural motif may be very similar to one another. However, it is possible for seemingly unrelated primary sequences to result in folding into a common structure motif

23. Structural and Functional Domains • Domains: Distinct regions of protein tertiary structure are often referred as domains • Three main classes ofprotein domains: structural domain, functional domain and topological domain • Functional domain: a region of a protein that exhibits a particular activity characteristic of the protein even when it is isolated from the rest of the protein • A structural domain is a region ~40 or more amino acids in length, arranged in a stable, distinct secondary or tertiary structure, that often fold into its characteristic structure independently of the rest of the protein • Topological domain: Distinctive special relationships with the rest of protein • 3o and 4o structure of hemagglutinin (HA), a surface protein of influenza virus • This multimeric molecule is made up of three identical submits, each composed of two polypeptide chains (HA1 and HA2) • The 4o structure of HA composed of 3 submits and the distal globular domain of each submitbinds sialic acid on the surface of the target cells

24. Denaturation and Renaturation of RibonucleaseA • Ribonuclease A is a single chain polypeptide. • Dr. Chris Anfinsen showed that denatuation of RNase A resulted in loosing the activity of the enzyme and re-naturation of the polypeptide regained the enzyme activity. • This discovery resulted in receiving a Nobel Prizes in 1973 (Assigned reading [I])

25. Hypothetical Protein-Folding Pathway (a). Primary structure (b)–(d). Secondary structure (e). Tertiary structure

26. Chaperonin-Mediated Protein Folding in E. coli • Prokartyotic GroEL in E. coli is a hollow barrel-shaped complex of 14 identical 60,000 MW submits arranged in two stacked rings • In the absence of ATP or presence of ADP, GeoEL esxist in a tight conformation state that binds partialy foldedd or misfolded proteins • Binding of ATP shifts GroEL to a more open relaxed state, which releases the folded protein • GroES, a co-chaperonin of 10,000 MW, helps the folding process

27. Eukaryotic Hsp70 Mediated Protein Assembly DnaJ/Hsp40 and GrpE/BAG1 are two accessory proteins involved in helping Hsp70 to promote the assembly of proteins Hsp70 in cytosol and mitochondrial matrix, BiP in endoplasmic reticulum, and DnaK in bacteria are molecular chaperones. Hsp70 and its homologs are major chaperones in all organisms

28. Quaternary Structure • Individual protein subunits interact between or among one another to form a complex entity • Hydrophobic or hydrophilic interaction between the side chains of amino acids in one submit with the side chains of amino acids in the other submit is responsible for formation of quaternary structure of a protein • The submits in the quaternary of proteins can be either identical submits or un-identical submits • With the formation of quaternary structure, proteins frequently quire additional functions

29. Aspartate Transcarbamoylase in E. coli • ATCase catalyzes the formation of N-carbamoylate from carbamoyl phosphate • This enzyme is a multimeric enzyme consists of catalytic submit (c=33 kD) and regulatory submit (r=17 kD) • The intact ATCase is 300 kD consists of c6r6 • C submit has catalytic activity alone. By combining with r submit, it assume allosteric effect • CTP has inhibit effect on ATCase and ATP has activation effect

30. Myoglobin and Hemoglobin • Myoglobin is a single chain polypeptide which can bind oxygen. • Hemoglobin consists of 2 a-globin chains and 2 b-globin chains. By forming the complete hemoglobin molecule, it assumes an allosteric effect

31. Assembly of Transcription Initiation Complex

32. Assembly of Tobacco Mosaic Virus

33. Assembly of T4 Phage

34. T4 Phage

35. Overview of Supramolecular Assembly of Macromolecules and the Biological Activities

36. Assigned Reading [I] • Nobel lecture by Chris Anfensen • Chaperonin-associated protein folding • Protein folding in the cell