N I V EST I G A T I O N O F T H E T H R E E -D I M E NSI O N A L S T R U C T U R E O F D N A
O B J E C T I V E : Students will use the S p a r t a n E S molecular modeling program to examine the structure of the DNA double helix, discover the structural properties of DNA bases, and identify the hydrogen bonds between them. The difference between the structures of one full turn of two different DNA conformations will be explored. B a c k g r ou n d : Nucleic acids (DNA and RNA) are the macromolecules that carry genetic information. The classical DNA is a pair of complementary strands of sugar-phosphate held together by specific inter-strand hydrogen bonding between the bases (adenine is paired with thymine, and guanine is paired with cytosine) and between the planes of base pairs.
F ig u r e 1. Base pairs (top) attached to the phosphate-sugar backbone (bottom). The bottom Figure was created by Madeleine Price Ball. It was obtained from the WikiMedia commons through which P e rmission is g r a nt ed to copy, d ist r ibut e a nd/o r modi fy th is document unde r the t e rms of the G N U F r e e D oc um e n t a t ion L i c e nse (se e h t t p : //e n . w i k i p e d i a .o r g/w i k i/ F i l e : D N A _c h e m i c a l_st r u c t u r e .sv g Last accessed January 2009.) [1]
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As shown below, the two strands are wound around each other to create the well-known double helix structure. This structure exhibits two structurally significant grooves: the socalled m i n o r a n d m a j o r g r oov es.
M a jor G r oo v e
M i no r G r oo v e
F ig u r e 2. Double helix structure of DNA is shown with the major and minor grooves indicated. This figure of the overview of the structure of DNA was created by Michael Strck February 8, 2006. Released under the GFDL (GNU Free Document License, http://en.wikipedia.org/wiki/GNU_Free_Documentation_License, last accessed January 2009). [1]
D N A co n f o r m a t i o n s Right-handed DNA (found in cells) can exist in two distinct double helix conformations. At high humidity (and low salt concentration) the favored form is called B-DNA and at low humidity (and high salt concentration) the dominant structure is called A-DNA. The bases in the B-form duplex (right side of the Figure 3) are virtually planar, and perpendicular to the helix axis. Each base pair rotates 36 relative to its neighbors. There are 10 base pairs per turn of a B-DNA helix. The B-DNA arrangement results in similar major and minor groove depths with the major groove width approximately twice the size of the minor groove width. 2
The base pairs of A-DNA (left side of Figure 3) are not perpendicular to the helical axis, but rather are tilted by about 20. A-DNA helices have 11 base pairs per turn. The diameter (width) of the A-DNA helix is approximately 6 ngstrms greater than that of B-DNA. The shape of the A-DNA duplex is characterized by the presence of a deep narrow major groove and a wide shallow minor groove. The A-DNA structure is similar to that found in double helical regions of RNA. This structural polymorphism is now accepted as local, sequence-dependent modulations of structure which are primarily associated with changes in the orientation of bases. Different base sequences have their own characteristic signature: they influence groove width, helical twist, curvature, mechanical rigidity, and resistance to bending. It seems probable that these features help proteins to read and recognize one base sequence in preference to another.
Figure 3. Structures of the A (left side) and B (right side) forms of DNA. Obtained in part from http://en.wikipedia.org/wiki/File:A-DNA,_B-DNA_and_Z-DNA.png. Released under the GFDL (GNU Free Document License, http://en.wikipedia.org/wiki/GNU_Free_Documentation_License, last accessed January 2009). [1]
G e t t i ng st a r t e d The following directions will lead you step-by-step through building and investigating the DNA molecule. To start the Spartan 06 ES molecular modeling program you must go to the Desktop and click on the green and yellow Spartan 06 ES icon (ES stands for ESsential edition. You will also see Spartan 06 (Full Edition) on your desktop, which should not be used). After the program is loaded, click on the Maximize icon in the upper-left hand corner to completely fill the screen with the Spartan window. Then go to F i l e and click N e w . Then click on the N u c l eot i d e tab on the right-hand side of the screen. This tab allows you to create models of nucleic acids. Pick D N A from the pull-down menu AGTC. Then go to the M od e l M e n u and pick B a l l a n d Sp o k e. This model allows viewing the
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locations of all atoms in a molecule. In order to distinguish atoms of different elements, atoms (represented as spheres) are colored in red for oxygen, black for carbon, blue for nitrogen, white for hydrogen and yellow for phosphorus. U se f u l co m m a n d s : Se l e c t - Click (left mouse button) on object. R ot a t e - Move mouse holding the left button. T r a n sl a t e - Move mouse with right button depressed. Sc a l e - Press sh i f t and move mouse pressing the right button. P a r t A : I n v es t i g a t i n g t h e d i f f e r e n c e b e t w e e n D N A b a ses a n d b a se p a i r s In order to understand the structure of a DNA molecule, first we are going to examine individual bases and the base pairs AT and GC (adenosine-thymine and guanine-cytosine base pairs respectively). These base pairs are the cross-sections of the double helix. The bases in the base pairs are called pyrimidines (T and C each having one ring) or purines (A and G each having two rings). The steps to use to examine the bases and base pairs follow. Fill in Part A of the data sheet for both the AT and GC base pairs. The instructions guide you through the study of AT. When finished with the AT base pair, you repeat the same steps for the GC base pair. 1. Select B-type helix of DNA. In order to bring bases onto the screen, click on the letter A button. The symbol (A-) will appear in the little white box. Now click once, using your left mouse button, in the big . This will bring to the screen Adenine (A) and the matching base thymine (T). The molecules may be rather large so you may need to use the useful commands (above) to make them smaller (Sc a l e) and move them to a convenient viewing perspective ( R ot a t e , T r a nsl a t e ). Examine each of the two bases carefully. Try to find all differences between each of the bases. Enter your observations into the Data Sheet. 2. How are these two bases bound to each other? To see it clearly go to M od e l and choose the H y d r oge n B on d s option. Now you can see exactly how these two bases are connecting to each other. Observe the number of hydrogen bonds which occur in A-T base pairing. Try to figure out which atoms are connected by hydrogen bonding, and distinguish between the donor and acceptors of the hydrogen bonds. G eo m e t r y and select M e asu r e D ist a n c e . Choose one end of the hydrogen bond and left-click, and then pick the other end. The measured distance should appear at the lower right hand corner of the screen, in units of ngstrms, or (1 =10-10 m). Measure all hydrogen bond distances and record the values on the data sheet and calculate the average. What conclusion can you draw about the hydrogen bonding distances in each type of base pair? What is the relevance of this for the structure of DNA? Given that the greater the number of Hydrogen bonds between bases is
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directly proportional to the overall stability of a base pair determine which set of base pairs is held more firmly together. 4. While you have your AT base pair displayed, pick out two atoms that are furthest apart and measure their distance. Make 2-3 choices of such atom pairs and calculate the average. This distance represents the diameter of double helix. 5. Go to E d i t and select C l e a r . AGTC buttons. Your screen now is ready to bring another base-pair for the investigation. 6. Repeat steps 1-5 for a cytosine and guanine base pair. Record observations your on the data sheet. P a r t B : E x a m i n i n g T w o B a se P a i r s 7a. Before looking into the full DNA structure, -dimensional structure of two base pairs, one pair stacked above the other. Remember that the base pairs are connected with sugar-phosphate backbones that hold the stacked bases together and provide the negative charges exposed to the surrounding solution to make DNA very soluble in water. In order to bring the two base pairs on the screen click base A and subsequently base G. This will give us the strand sequence of AG. When you click on the green View window, you will see these connected to one another as well as the corresponding matching bases in the sequence; Adenine (A) and matching base thymine (T), guanine (G) with matching base cytosine (C). The result is a two step mini-helix. The perpendicular distance between the two parallel base pair planes is called the rise. Turn on the H y d r oge n B on d s option if it is not still active. Examine each base carefully. Look to the right of the Spartan screen. There you will find the value for the rise. Record this value on your data sheet. Another important property of the DNA molecule is the t w ist. The twist is the degrees that one base pair plane is rotated with respect to the other. Record the twist value for this DNA mini helix on your data sheet. Also record the value of the twist on your data sheet. Complete the calculation of the height of a complete turn and the number of base pairs in a complete turn. 7b. Repeat the above instructions but this time choose the A-DNA form. Notice that while the resulting model is still an A-G mini-helix its shape is a bit distorted. Look at the rise and twist value. Record these on the data sheet and complete the calculation for number of bases per complete turn and the height of a complete turn of A-DNA P a r t C : E x a m i n i ng a f u l l D N A d u p l e x f r agme n t structure of the 3-dimensional helix by studying a dodecamer (12 base pair steps) duplex fragment. Clear the screen, and select the B type helix radio button. Use the A G T C buttons to enter the sequence C-G-C-G-A-A-T-T-C-G-C-G (the required connecting bonds will automatically be included). This sequence is one of the most widely investigated sequences of DNA. Next click the green View screen to see the
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duplex. Go to F i l e and S a v e BClose the file. Next, build the same sequence but select the A-type helix of DNA. Go to F i l e and S a v e A9. Re-open the first file Band keep both structures open on the screen, moving them as necessary for side-by-side comparison. Click on the structure you want to examine and manipulate as necessary to answer the questions that follow. As you examine the DNA structure you will see that there are some pronounced differences in the helical structures. For both types measure the r ise of the helix, the height of a full turn and the number of base pairs per turn. You might use the data you obtained earlier in this study to help you. Locate the major and minor grooves in both DNA forms. It may be helpful to use the Space Filling models (go to the M od e l M e n u ) to determine the differences between the helix depths and sizes. Try using the Tube model with the Ribbons option turned on to view the helices more clearly. Afterwards, rotate the duplex so the helix axis is perpendicular to the screen and examine the cross section of the helix as you look down the helix axis. Measure the diameter of both DNA structures. Write a statement identifying a key observation when comparing A- and B- forms of DNA helices observed down the helix axis. Record your observations on the data sheet in section Part C. 10. When you are finished exit the Spartan program and please d you are through with your work for the day. R e f e r e n c es : [1] http://en.wikipedia.org/wiki/DNA . Last accessed January 2009. [2] D.D. Ebbing and S.D. Gammon, General Chemistry (9th ed., Houghton Mifflin, New York), 2005. See 11.5 (pages 440-442 for hydrogen bonding) and 25.4 (pages 1017-1022 for Nucleic Acids). [3] L. Stryer, Biochemistry (2nd edition, W.H. Freeman & Co., San Francisco), 1981. O t h e r U se f u l L i n k s : http://www.whatislife.com/reader/dna-rna/dna-rna.html (Last accessed January 2010) http://tigger.uic.edu/classes/phys/phys461/phys450/ANJUM04/ (Last accessed January 2010)
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Name ____________________ D A T A SH E E T
Date __________________ 3 D St r u c t u r e o f D N A
P a r t A : I n v es t i g a t i n g t h e d i f f e r e n c e b e t w e e n D N A b a ses a n d b a se p a i r s
A adenine How many carbon rings are there in each base? T thymine G guanine C cytosine
Check if the following functional groups are present: - NH - C=O - NH2 - CH3
Is this base a purine?
Is this base a pyrimidine?
The base with the -CH3 functional group is _______________________ . The base with the -NH2 group but without the C=O group is ________________ . A purine with the -C=O group is __________________________ . A pyrimidine with the -NH2 group is ________________ .
How many hydrogen bonds hold together Adenine and Thymine? Guanine and Cytosine? ____________ ____________ A-T or G-C
W h i c h b ase p a i r s a r e most st r on gl y b on d e d (c i r c l e)?
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List all hydrogen bond connections of form (base)__O H N__ (base) or (base) __N H .N__ (base) A-T base pair G-C base pair ________________________________________________ ________________________________________________
What is the average distance between hydrogen bonded atoms for each base? (obtain three distances between N H or H O atoms) #1 Adenine Thymine Guanine Cytosine ______ ______ #2 ______ ______ ______ #3 average ________ ________
T h e a v e r age l e ng t h of H y d r oge n B on d ___________________ (Average all distances to obtain an overall average) Compare a hydrogen bond length to an average chemical bond length.
Compare the energy of a hydrogen bond to an average chemical bond.
Measure distances between the two furthest atoms three times using different atoms as reference points. This measurement gives you an approximate width of the helix. #1 Adenine Thymine Guanine Cytosine ______ ______ #2 ______ ______ #3 ______ ______ average ________ ________
T h e w i d t h of t h e h e l i x _________________________ (Average all six distances to obtain an overall average width) 8
P a r t B : E x a m i n i n g T w o B a se P a i r s
B - D N A - The average distance between the planes of the two stacked base pairs is called the base pair r ise.
T h e r i se o f t h e B - D N A h e l i x i s T h e B - D N A H e l i x T w i st i s _________________ _________________
One complete turn of the helix is 360. Compute t h e n u m b e r of b ase p a i r p l a n es f o r on e co m p l e t e t u r n o f t h i s B - D N A . ____________________________ Given the rise for a pair of base planes, compute t h e tot a l h e igh t of a f u l l t u r n of B - D N A . _____________________________
A - D N A - The average distance between the planes of the two stacked base pairs called the base pair r ise. T h e r i se o f t h e A - D N A h e l i x i s _________________
A - D N A H e l i x T w ist _________________
One complete turn of the helix is 360. Calculate t h e n u m b e r of b ase p a i r p l a n es f o r on e co m p l e t e t u r n o f t h i s A - D N A . __________________________________ Given the rise for a pair of base planes, calculate the total h e igh t of a f u l l t u r n of A - D N A . ___________________________________ P a r t C : C o m p a r i n g t h e co n f o r m a t i o n o f t h e f u l l D N A d u p l e x f r a g m e n t s A -D N A the width of the helix the rise of the helix the height of one full turn # of base pair steps per full turn ___________ ___________ ___________ ___________ B-D N A ____________ ____________ ____________ ____________
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V i e w i n g d ow n t h e h e l i x a x es s t a t e m e n t ( a co m p l e t e se n t e n c e) .
P a r a g r a p h c o n t r a s t i n g t h e t w o f o r ms o f D N A .
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