Lecture 2 Molecular Structure of DNA and RNA part 2 Chapter 9, pages

9-31 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display In 1953, James Watson and Francis Crick discovered the double helical structure of DNA The scientific framework for their breakthrough was provided by other scientists including Linus Pauling Rosalind Franklin and Maurice Wilkins Erwin Chargaff A Few Key Events Led to the Discovery of the Structure of DNA

9-32 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display In the early 1950s, he proposed that regions of protein can fold into a secondary structure  -helix Linus Pauling Figure 9.12 To elucidate this structure, he built ball-and-stick models Refer to Figure 9.12b

9-33 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display She worked in the same laboratory as Maurice Wilkins She used X-ray diffraction to study wet fibers of DNA Rosalind Franklin The diffraction pattern is interpreted (using mathematical theory) This can ultimately provide information concerning the structure of the molecule

9-34 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display She made marked advances in X-ray diffraction techniques with DNA The diffraction pattern she obtained suggested several structural features of DNA Helical More than one strand 10 base pairs per complete turn Rosalind Franklin

9-35 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Chargaff pioneered many of the biochemical techniques for the isolation, purification and measurement of nucleic acids from living cells It was already known then that DNA contained the four bases: A, G, C and T Erwin Chargaff’s Experiment

The Hypothesis An analysis of the base composition of DNA in different species may reveal important features about the structure of DNA 9-36 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Testing the Hypothesis Refer to Figure 9.14

9-37 Figure 9.14

9-38 Figure 9.14

The Data 9-39 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Interpreting the Data 9-40 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The data shown in Figure 9.14 are only a small sampling of Chargaff’s results The compelling observation was that Percent of adenine = percent of thymine Percent of cytosine = percent of guanine This observation became known as Chargaff’s rule It was crucial evidence that Watson and Crick used to elucidate the structure of DNA

9-41 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Familiar with all of these key observations, Watson and Crick set out to solve the structure of DNA They tried to build ball-and-stick models that incorporated all known experimental observations A critical question was how the two (or more strands) would interact An early hypothesis proposed that the strands interact through phosphate-Mg ++ crosslinks Refer to Figure 9.15 Watson and Crick

9-42 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Figure 9.15 This hypothesis was, of course, incorrect!

9-43 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display They went back to the ball-and-stick units They then built models with the Sugar-phosphate backbone on the outside Bases projecting toward each other They first considered a structure in which bases form H bonds with identical bases in the opposite strand ie., A to A, T to T, C to C, and G to G Model building revealed that this also was incorrect Watson and Crick

9-44 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display They then realized that the hydrogen bonding of A and T resembled that between C and G So they built ball-and-stick models with AT and CG interactions These were consistent with all known data about DNA structure Refer to Figure 9.16 Watson, Crick and Maurice Wilkins were awarded the Nobel Prize in 1962 Rosalind Franklin died in 1958, and Nobel prizes are not awarded posthumously Watson and Crick

9-45 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display General structural features (Figures 9.17 & 9.18) The DNA Double Helix Two strands are twisted together around a common axis There are 10 bases per complete twist The two strands are antiparallel One runs in the 5’ to 3’ direction and the other 3’ to 5’ The helix is right-handed As it spirals away from you, the helix turns in a clockwise direction

9-46 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display General structural features (Figures 9.17 & 9.18) The DNA Double Helix The double-bonded structure is stabilized by 1. Hydrogen bonding between complementary bases A bonded to T by two hydrogen bonds C bonded to G by three hydrogen bonds 2. Base stacking Within the DNA, the bases are oriented so that the flattened regions are facing each other

9-47 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display General structural features (Figures 9.17 & 9.18) The DNA Double Helix There are two asymmetrical grooves on the outside of the helix 1. Major groove 2. Minor groove Certain proteins can bind within these grooves They can thus interact with a particular sequence of bases

9-48 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Figure 9.17

9-49 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Figure 9.18

9-50 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The DNA double helix can form different types of secondary structure The predominant form found in living cells is B-DNA However, under certain in vitro conditions, A-DNA and Z-DNA double helices can form DNA Can Form Alternative Types of Double Helices

9-51 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display A-DNA Right-handed helix 11 bp per turn Occurs under conditions of low humidity Little evidence to suggest that it is biologically important Z-DNA Left-handed helix 12 bp per turn Its formation is favored by GG-rich sequences, at high salt concentrations Cytosine methylation, at low salt concentrations Evidence from yeast suggests that it may play a role in transcription and recombination

9-52 Figure 9.19 Bases substantially tilted relative to the central axis Sugar-phosphate backbone follows a zigzag pattern Bases relatively perpendicular to the central axis

9-53 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display In the late 1950s, Alexander Rich et al discovered triplex DNA It was formed in vitro using DNA pieces that were made synthetically In the 1980s, it was discovered that natural double- stranded DNA can join with a synthetic strand of DNA to form triplex DNA The synthetic strand binds to the major groove of the naturally-occurring double-stranded DNA Refer to Figure 9.20 DNA Can Form a Triple Helix

9-54 Figure 9.20 Triplex DNA formation is sequence specific The pairing rules are Triplex DNA has been implicated in several cellular processes Replication, transcription, recombination Cellular proteins that specifically recognize triplex DNA have been recently discovered T binds to an AT pair in biological DNA C binds to a CG pair in biological DNA

9-55 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display To fit within a living cell, the DNA double helix must be extensively compacted into a 3-D conformation This is aided by DNA-binding proteins Refer to 9.21 This topic will be discussed in detail in Chapter 10 The Three-Dimensional Structure of DNA

9-56 Figure 9.21 DNA wound around histone proteins

9-57 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The primary structure of an RNA strand is much like that of a DNA strand Refer to Figure 9.22 vs RNA strands are typically several hundred to several thousand nucleotides in length In RNA synthesis, only one of the two strands of DNA is used as a template RNA Structure

9-58 Figure 9.22

9-59 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Although usually single-stranded, RNA molecules can form short double-stranded regions This secondary structure is due to complementary base- pairing A to U and C to G This allows short regions to form a double helix RNA double helices typically Are right-handed Have the A form with 11 to 12 base pairs per turn Different types of RNA secondary structures are possible Refer to Figure 9.23

9-60 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Figure 9.23 Also called hair-pin Complementary regions Noncomplementary regions Held together by hydrogen bonds Have bases projecting away from double stranded regions

9-61 Many factors contribute to the tertiary structure of RNA For example Base-pairing and base stacking within the RNA itself Interactions with ions, small molecules and large proteins Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Figure 9.24 depicts the tertiary structure of tRNA phe The transfer RNA that carries phenylalanine Molecule contains single- and double- stranded regions These spontaneously interact to produce this 3-D structure Figure 9.24