Introduction to molecular. Dr Saeb Aliwaini

Transcription

1 Introduction to molecular biology Dr Saeb Aliwaini

2 Course description The principal aim of the course is to equip students with a basic knowledge of the molecular biology of the cells

3 Course description Basic properties of cells, techniques used in cell and molecular biology, the structure and function of the nucleus, Genes and chromosomes, DNA replication, transcription, translation, cell signalling. This will be linked to diseases and its diagnosis and treatment.

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5 Course description For example, what is cancer how do we diagnose and treat.

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10 Week topic 1 The Molecular Nature of Genes : 2 Gene Function 3 From gene to protein 4 DNA replication and telomere maintenance 5 Cell cycle control, apoptosis, autophagy 6 DNA repair and recombination 7 Cancer 8 Molecular diagnostics 9 Molecular Therapeutics

11 Grades: Midterm Exams 30% Final Exam 60% Quizzes 10% Total = 100 %

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13 DNA and RNA The Nature of Genetic Material RNA is composed of a sugar (ribose) plus four nitrogencontaining bases, and that DNA contains a different sugar (deoxyribose) plus four bases. Each base is coupled with a sugar phosphate to form a nucleotide Streptococcus pneumoniae A spherical cell surrounded by a mucous coat called a capsule

14 Somehow the virulent trait passed from the dead cells to the live, avirulent ones. The missing piece of the puzzle was the chemical nature of the transforming substance

15 Genes are made from DNA Later it was found that the enzyme deoxyribonuclease (DNase), which breaks down DNA, destroyed the transforming ability of the virulent cell extract. These results suggested that the transforming substance was DNA. Confirmed by: Ultracentrifugation Electrophoresis Ultraviolet Absorption Spectrophotometry

16 Genes are made from DNA Yet, by 1953, when James Watson and Francis Crick published the double-helical model of DNA structure, most geneticists agreed that genes were made of DNA. Bacteriophage (bacterial virus) called T2 that infects the bacterium Escherichia coli. The phage genes enter the host cell and direct the synthesis of new phage particles

17 Genes are made from DNA The phage is composed of protein and DNA only. The question is this: Do the genes reside in the protein or in the DNA? on infection, most of the DNA entered the bacterium, along with only a little protein. The bulk of the protein stayed on the outside Other experiments showed that some viral genes consist of RNA.

18 The Chemical Nature of Polynucleotides bases, phosphoric acid, and the sugar deoxyribose (hence the name deoxyribonucleic acid). The four bases found in DNA are adenine (A), cytosine (C), guanine (G), and thymine (T). Adenine and guanine are related to the parent molecule, purine.

19 Primes are used in the numbering of the ring positions in the sugars to differentiate them from the ring positions of the bases. Hydrogen and carbon atoms are usually omitted for clarity.

20 Nitrogenous bases Nitrogen-containing molecules having the chemical properties of a base (a substance that accepts an H + ion or proton in solution). Chargaff s rules: - Using chromatography that - [A]=[T] conc - [G]=[C] conc - And purine bases equals that of the pyrimidine bases ([A]+[G]=[T]+[C]) - percent G+C, differs among species but is constant in all cells of an organism within a species. The G+C content can vary from 22 to 73%, depending on the organism.

21 Sugars - RNA contains a pentose (five-carbon) sugar called ribose. - DNA contain the sugar deoxyribose. - Both sugars have an oxygen as a member of the five-member ring. - The 5 -carbon is outside the ring. - Sugars differ only in the presence or absence ( deoxy ) of an oxygen in the 2 position.(2 -hydroxyl group) The base is linked to the 1 -position of the sugar= nucleoside Nucleotides are nucleosides with a phosphate group attached through a phosphoester bond

22 The phosphate functional group (PO 4 ) gives DNA and RNA the property of an acid (a substance that releases an H + ion or proton in solution) nucleic acid. esters linking give stabilization after the phosphodiester bond is formed, one oxygen atom of the phosphate group is still negatively ionized The negatively charged phosphates are extremely insoluble in lipids. How does this help? An ester is an organic compound formed from an alcohol (bearing a hydroxyl group) and an acid

23 These are called phosphodiester bonds because they involve phosphoric acid linked to two sugars: one through a sugar 5 -group, the other through a sugar 3 -group The top of the molecule bears a free 5 -phosphate group, so it is called the 5 -end. The bottom, with a free 39- hydroxyl group, is called the 3 - end

24 Significance of 5 and 3 This 5 3 directionality of a nucleic acid strand is an extremely important property of the molecule. By convention, a DNA sequence is written with the 5 end to the left, and the 3 end to the right. The number of base pairs (bp) is used as a measure of length of a doublestranded DNA. kilobase pair (kb or kbp) OR (Mb or Mbp) (usually less than 50 bases) called oligonucleotides. This lecture will focus on DNA. Various chemical forces drive the formation of the DNA double helix. These include hydrogen bonds between the bases and base stacking by hydrophobic interactions.

25 Hydrogen bonds Between the nitrogenous bases on opposite strands of the interwound DNA chains.

26 Hydrogen bonds are very weak bonds that involve the sharing of a hydrogen between two electronegative atoms, such as oxygen and nitrogen. The hydrogen bonding between bases is referred to as Watson Crick or complementary base pairing. adenine (A) normally pairs with thymine (T) by two hydrogen bonds, and guanine (G) pairs with cytosine (C) by three hydrogen bonds.

27 There are proofreading mechanisms and DNA repair mechanisms that recognize non Watson Crick base pairs and correct the majority of mistakes G-U base pairing is stable, and is of importance in RNA structure and RNA protein interactions

28 Base stacking provides chemical stability to the DNA double helix The nitrogenous bases are hydrophobic nonpolar. Once the bases are attached to a sugar and a phosphate to form a nucleotide, they become soluble in water, but even so their insolubility still places strong constraints on the overall conformation of DNA in solution. The paired, relatively flat bases tend to stack on top of one another by means of a helical twist

29 A double-stranded DNA molecule thus has a hydrophobic core composed of stacked bases, the sugars and phosphates are soluble in water they orient towards the outside of the helix.

30 Structure of the Watson Crick DNA double helix polarity in each strand (5 3 ) of the DNA double helix: one end of a DNA strand will have a 5 -phosphate and the other end will have a 3 -hydroxyl group Watson and Crick found that hydrogen bonding could only occur if the polarity of the two strands ran in opposite directions The DNA double helix is also referred to as double-stranded DNA (dsdna) or duplex DNA to distinguish it from the single-stranded DNA (ssdna) found in some viruses

31 DNA has a structure very similar to the one just described, but the helix contains about 10.4 bp per turn.

32 The sugar phosphate backbone is not equally spaced and results in what are called the major and minor grooves of DNA. Two grooves of different width a wider major groove and a more narrow minor groove that spiral around the outer surface of the double helix. The major groove carries a message (the base sequence of the DNA) in a form that can be read by DNA-binding proteins. most transcription factors (proteins involved in regulating gene expression) bind DNA in the major groove.

33 B-DNA is a right-handed helix; it turns in a clockwise manner when viewed down its axis B-DNA occurs under conditions of high humidity (95%) and relatively low salt The predominant form in vivo is B-DNA. If the water content is decreased and the salt concentration increased (A-DNA) will occur the bases are tilted with respect to the axis and thereare more (11) bases per turn than in B-DNA backbone formed a zig-zag structure, they called the structure Z-DNA A left-handed helix turns counterclockwise

34 DNA can undergo reversible strand separation During DNA replication and transcription, the strands of the helix must separate transiently and reversibly. This equals denaturation in Vitro = unwinding Heating lead to broken hydrogen bonds, whereas the phosphodiester bonds remain intact. As DNA denatures, its absorption of UV light increases, a phenomenon known as hyperchromicity. The temperature at which half the bases in a double-stranded DNA sample have denatured is denoted the melting temperature (Tm)

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37 Hyperchromic shift The amount of strand separation, or melting, is measured by the absorbance of the DNA solution at 260 nm. Nucleic acids absorb light at this wavelength because of the electronic structure in their bases. But when two strands of DNA come together, the close proximity of the bases in the two strands quenches some of this absorbance. When the two strands separate, this quenching disappears and the absorbance rises 30 40%

38 Heating is not the only way to denature DNA. Organic solvents such as dimethyl sulfoxide and formamide, or high ph, disrupt the hydrogen bonding between DNA strands and promote denaturation. When heated solutions of denatured DNA are slowly cooled, single strands often meet their complementary strands and form a new double helix. This is called renaturation or annealing. Lowering the salt concentration of the DNA solution also aids denaturation by removing the ions that shield the negative charges on the two strands from each other. At very low ionic strength, the mutually repulsive forces of these negative charges are strong enough to denature the DNA at a relatively low temperature.

39 The G+C content of a DNA molecule has a significant effect on its Tm because GC base pair has three hydrogen bonds

40 The melting temperature also depends on the salt concentration: in low salt, a given DNA will melt at a lower temperature than in a higher salt concentration. This is because DNA is a polyanionic molecule. The salt "shields" the negative charges on each phosphate. When the charges are NOT shielded, the electrostatic repulsion makes it energetically more favorable to separate the strands.

41 Also high ph or organic solvents such as formamide disrupt Also high ph or organic solvents such as formamide disrupt the hydrogen bonding between DNA strands and promote denaturation.

42 Unusual DNA secondary structures Slipped structures : 5 -TACGTACGTACGTACG-3 A tandem repeat (sometimes called a direct repeat) usually upstream of regulatory sequences Cause DNA to assume unusual DNA secondary structures and blocking replication forks and promoting repair Friedreich s ataxia The disorder is caused by a 5 -GAA-3 trinucleotide repeat expansion in the first intron of the Friedreich s ataxia gene, which is located on chromosome 9 the earlier the onset of the disease and the quicker the decline of the patient.

43 Friedreich s ataxia Normally, the gene may be expanded up to ~33 repeats (GAA trinucleotide repeats), but when the sequence is longer than 59 repeats it appears to alter the architecture of the DNA sequence by causing the usual double-helix structure to fold back on itself into a triplex formation ( sticky DNA') (Sakamoto et al 1999). This interferes with transcription of the gene, resulting in a deficiency of the protein encoded in that gene, in this case named frataxin (Bidichandani SI, et al 1998). Increasing length of the repeat expansion correlates with greater reduction in levels of frataxin and increased severity of the disorder (Campuzano et al 1997).

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45 Cruciform structures Experimental evidence has led to the hypothesis that cruciform structures can act as regulatory elements in DNA replication and gene expression in various prokaryotic and eukaryotic systems. Confirmation of a functional role in vivo awaits further investigation. The DNA becomes rearranged so each repeat pairs with the complementary sequence on its own strand of DNA, instead of with the complement on the other strand. Triple helix DNA

46 Tertiary structure of DNA Many naturally occurring DNA molecules are circular, with no free 5 or 3 end. The 5 end of one strand can only join its own 3 end to covalently close a circle. Two circles of single-stranded DNA twisted around each other This DNA can then become supercoiled, supercoils are a twisted, three-dimensional structure which is more favorable energetically

47 linking number (L) of 10 the result is an energetically relaxed circle that lies flat underwound by one full turn to the left and then the ends are sealed together, If L=9 the result is a strained circle

48 Generally, changes in the average number of base pairs per turn of the double helix will be counteracted by the formation of an appropriate number of supercoils in the opposite direction Overtwisting of the double helix usually leads to positive (right-handed) one negative (left-handed)

49 For example, if the double helix is overwound by one full turn to the right and then the ends are sealed together the result is a strained circle with 9.5 bp/turn. The supercoiled state is inherently less stable than relaxed DNA The stress present within supercoiled DNA molecules sometimes leads to localized denaturation After replication and transcription

50 Topoisomerases relax supercoiled DNA Topological isomers (topoisomers) Topoisomers can be visualized by their differing mobilities when separated by gel electrophoresis Both type I and type II topoisomerases play important roles in many cellular processes, including chromosome condensation and segregation, DNA replication, gene transcription, and recombination

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52 Topoisomerases are highly conserved enzymes that convert (isomerize) one topoisomer of DNA to another type I and type II Type I topoisomerases are proficient at relaxing supercoiled DNA They act by forming a transient single-stranded break in the DNA (cleavage of a phosphodiester bond between adjacent nucleotides) and, while winding the broken ends, pass the other strand through the break

53 Topoisomerase-targeted anticancer drugs