分子生物学Chapter 3 Nucleic Acids and Genomics.docx

1、分子生物学Chapter 3 Nucleic Acids and GenomicsChapter 3 Nucleic Acids and Genomics Review of nucleic acidsThe structure of DNA, Deoxyribonucleic acid, was first published in the British journal Nature in 1953 by J.D. Watson and F.H. Crick. However, the constituents of DNA had been known since the turn of

2、 the century. The simple model proposed by Watson and Crick implemented X-ray crystallography performed by M.H.F. Wilkins and R. Franklin. Additionally, work by L. Pauling provided the rules of bonding and the elucidation of the alpha helix structure. E. Chargraff had provided the data on the abunda

3、nce of the four nucleotides (Adenine, Thymine, Guanine, and Cytosine) and the relationships between them from chemical analysis. E. Chargraff determined that distribution of A and T were proportional, and that distribution of G and C were also proportional suggesting the complementary arrangement of

4、 adenine-thymine and guanine-cytosine. Since the arrangement of DNA was complementary, the sequence of one chain must be compatible to the sequence of the opposite chain. Francis Crick (left), Jim Watson (centre), and Linus Pauling (right). Images from the Nobel Prize Foundation web site Maurice Wil

5、kins showed a diffraction pattern of DNA at a scientific meeting in Naples in 1951. This was the first diffraction pattern that Watson saw of DNA and it had a decisive impact in his decision to study DNA. Watson moved to the Cavendish laboratory in Cambridge where he struck up his famous collaborati

6、on with Francis Crick.Their first attempt at a model structure, late in 1951, was wrong - embarassingly so for them at the time, since they had arranged for Maurice Wilkins and Rosalind Franklin to travel up from London to view their structure. Their model was a triple helix in which the polynucleot

7、ide backbones were placed at the centre of the structure with the bases pointing out in solution. But, this arrangement of chains was chemically impossible and once this was pointed out, the model fell apart.X-Ray fibre diffraction patters of A-DNA (left) and B-DNA (right). Images from the Maurice W

8、ilkins 1952 Nobel Lecture at the Nobel Prize Foundation web siteChapter 3 Nucleic Acids and Genomics 1. Building Blocks Nucleotidesv A nucleotide is composed of three parts: pentose, base and phosphate group. v A, C, G and T exist in DNA;v A, C, G and U exist in RNA. Bases:Bicyclic Purines:Monocycli

9、c pyrimidine:Nucleosides:The bases are covalently attached to the 1 position of a pentose sugar ring, to form a nucleosideNucleotides:A nucleotide is a nucleoside with one or more phosphate groups bound covalently to the 3-, 5, or ( in ribonucleotides only) the 2-position. In the case of 5-position,

10、 up to three phosphates may be attached. BASESNUCLEOSIDESNUCLEOTIDESAdenine (A)AdenosineAdenosine 5-triphosphate (ATP)DeoxyadenosineDeoxyadenosine 5-triphosphate (dATP)Guanine (G)GuanosineGuanosine 5-triphosphate (GTP)Deoxy-guanosineDeoxy-guanosine 5-triphosphate (dGTP)Cytosine (C)CytidineCytidine 5

11、-triphosphate (CTP)Deoxy-cytidineDeoxy-cytidine 5-triphosphate (dCTP)Uracil (U)UridineUridine 5-triphosphate (UTP)Thymine (T)Thymidine/deoxythymidieThymidine/deoxythymidie 5-triphosphate (dTTP)Phosphodiester bonds & primary sequence:Primary sequence:5end: not always has attached phosphate groups3 en

12、d: free hydroxyl (-OH) group 2. DNA StructureWatson and Crick , 1953The genetic material of all organisms except for some virusesThe foundation of the molecular biologyBase pairingTwo separate strands Antiparellel (53 direction)Complementary (sequence)Base pairing: hydrogen bonding that holds two st

13、rands together Sugar-phosphate backbones (negatively charged): outside Planner bases (stack one above the other): inside2.1 DNAs B Form, A Form and Z Formv In this structure, also known as the B form, the helix makes a turn every 3.4 nm, and the distance between two neighboring base pairs is 0.34 nm

14、. Hence, there are about 10 pairs per turn. The intertwined strands make two grooves of different widths, referred to as the major groove and the minor groove, which may facilitate binding with specific proteins.v The normal right-handed double helix structure of DNA, also known as the B formv In a

15、solution with higher salt concentrations or with alcohol added, the DNA structure may change to an A form, which is still right-handed, but every 2.3 nm makes a turn and there are 11 base pairs per turn. Features of the Watson-Crick model of B-DNA：v It is an antiparallel double helix.v It is a right

16、-handed helix.v The base-pairs are perpendicular to the axis of the helix. (Actually, they are very slightly tilted - at an angle of 4 degrees)v The axis of the helix passes through the centre of the base pairs.v Each base pair is rotated by 36 degrees from the adjacent base pair.v The base-pairs ar

17、e stacked 0.34 nm apart from one another.v The double helix repeats every 3.4 nm, i.e. the pitch of the double helix is 3.4 nm.v B-DNA has two distinct grooves: a MAJOR groove; and, a MINOR groove. These grooves form as a consequence of the fact that the beta-glycosidic bonds of the two bases in eac

18、h base pair are attached on the same edge. However, because the axis of the helix passes through the centre of the base pairs, both grooves are similar in depth.The real structure of B-DNAWatson and Cricks structure was a just a model - but a pretty good one. Nevertheless, it took nearly 30 years be

19、fore the structures of DNA were resolved at atomic resolution.In 1980, Richard Dickerson and Horace Drew solved the structure of a 12-mer double-stranded self-complementary oligonucleotide with the following sequence:5-CGCGAATTCGCG-3Their results showed that crystals of B-DNA had a structure very si

20、milar to that proposed by Watson and Crick. Although there were numerous small variations, the overall structure was as expected. The molecule was a right-handed double-helix. The backbone chains were antiparallel. The base pairs were very nearly perpendicular to the helix axis. The base pairs were

21、centred on the helix axis. On average each base pair was rotated 35.6 degrees from the adjacent base pair. However, the individual measured rotations (twist) varied from as little as 28 degrees to as much as 42 degrees.On average the rise per base pair (i.e. the distance between adjacent base pairs)

22、 was 0.34 nm. However, the rise between individual base pairs varied from 0.274 nm to 0.435 nm.One of the most striking features of the structure solved by Dickerson and Drew was that there is considerable variation in the structure of individual base pairs. Many were were not exactly planar but wer

23、e slightly twisted (propellor twist). This feature, as well as the variations in the twist angles and the rise between base pairs can be explained or understood as resulting from the complex interplay of attractive and repulsive forces due to the different chemical properties of the four distinct ba

24、ses within a DNA double helix. The detailed structure of a DNA molecule is actually influenced by the sequence.A-DNA Recall that the first fibre diffraction pictures taken by Rosalind Franklin were of a dehydrated form of DNA, which we now know as A-DNA. An easy way to remember the basic structure o

25、f A-DNA is to remember this fact about its water content. In A-DNA there is no water spine. The base pairs - which nevertheless are formed with canonical Watson-Crick hydrogen bonds - are pushed towards the minor groove. In doing so, the base pairs tilt to 19 degrees from perpendicular to the helix

26、axis which no longer passes through the centre of each base pair. The resulting minor groove is about as wide as the resulting major groove. However, the major groove is very deep while the minor groove is very shallow. In addition, the sugar ring changes from the C2-endo conformation found in B-DNA

27、 to a C3-endo conformation. This change serves to distance the phosphate from the C2 hydroxyl atom hence preventing autocatalysisThe following images show the structure of A-DNA. Note the following about these images: The first image shows the A-DNA helix from the side. Notice that the base pairs ar

28、e tilted with respect to the axis of the helix. Most of the base pairs are nearly planar, but, as with B-DNA, you can see some exceptions. You can also see some indication of propellor twist. Observe the base pair that is halfway along the helix. Your view of this base pair is essentially end-on. Fr

29、om this perspective, you can see that this base pair is not centred on the helix axis but is very much pushed to one side. The major groove (on the right) is very deep; the minor groove (on the left) is very shallow. The second image shows the A-DNA helix from one end. Notice that there is essential

30、ly a large hole in the centre because all of the atoms of the base pairs are pushed out to the sides. Another DNA structure is called the Z form, because its bases seem to zigzag. Z DNA is left-handed. One turn spans 4.6 nm, comprising 12 base pairs. v The DNA molecule with alternating G-C sequences

31、 in alcohol or high salt solution tends to have such structure. Z-DNA Ever since Watson and Crick proposed their structure for DNA, crystallographic proof was sought. In 1979, Alex Rich and his colleagues at MIT thought they had it! They succeeded in crystallizing the self-complementary hexanucleoti

32、de, CGCGCG. Their analysis was a surprise. Their crystals showed that this particular molecule adopted a left-handed double helix under their crystallization conditions. The bases had adopted a syn- conformation rather than the usual anti- conformation with the result that the repeating unit of the structure was a dinucleotide base