Issued as Final report, Project no. G-41-64

Wartell, Roger M.

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Scholarly Materials And Research @ Georgia Tech

GEORGIA INSTITUTE OF TECHNOLOGY Office of Contract Administration SPONSORED PROJECT INITIATION Date March 17, 1976 ...Project Titles The Transmission of Conforrnational Stability Throl ,gh Adjacent Regions of DNA Project No: G41-647.. Project Director: Dr. Rcger M. Wartell sponsor: National Science Foundation Agreement Period: From. '341./16 	Until 12 month budget period plus b ricntns ror surnitSTIDn of regritrel -M7MT:67—etc7----- Type Agreement: Grant No. PC M76-04565 Amount: $16,000 NSF (G-41-647) 4,530 GIT (G-41-338) $20,530 Total Reports Required: Annual Letter Technical, Final Report Sponsor Contact Person(s): Technical Matters  Contractual Matters  (thru OCA) Mr. Gaylord L. Ellis Grants Officer National Science Foundation Washington, DC '20550 (202) 632-5965 Assigned to:  physics 	 (School/Laboratory) Copies to: Project Director Division Chief (EES) School/Laboratory Director Dean/Director-EES Acco:Inting Cffic Procurement Office Security .Coordinator (OCA) Reports Coordinator (OCA) ,.. A -3 (3/76) Library, Technical Reports Section Office of Computing Services Director, Physical Plant EES Information Office Project File (C ='.) Project Code (GTRI) Other GEORGIA INSTITUTE OF TECHNOLOGY OFFICE OF CONTRACT ADMINISTRATION SPONSORED PROJECT TERMINATION Date: 	1/14/81 Project Title: 	Transmission of Conformational Stability Through Adjacent Regions of DNA Project No: 	G-41-647 Project Director: Dr. Roger M. Wartell Sponsor: 	National Science Foundation Effective Termination Date: 	May 31, 1980 Clearance of Accounting Charges: 	  Grant/Contract Closeout Actions Remaining: Final Invoice and Closing Documents — X Final Fiscal 13ergog Accounting (FCTR) X Final Report of Inventions (if positive) Govt. Property Inventory & Related Certificate Classified Material Certificate Other Physics Assigned to: 	  (School/XX)arliKX39 COPIES TO: Project Director Division Chief CEES) School/Laboratory Director Dean/Director—EES Accounting Office Procurement Office S9curity Coordinator COCA) /Reports Coordinator COCA) Library, Technical Reports Section EES Information Office Project File MCA/ Project Code MTRO Other OCA Research Property Coordinator  Project Code (OCA) CA-4 (1/79) r N„,:..: NA L SCIENCE FOUNDATION Washington, D.C. 20550 	 FINAL PROJECT REPORT NSF FORM 9XA 	 • PLEASE READ INSTRUCTIONS 0,V REVERSE PEFORE COMPLETING 	 • PART I—PROJECT IDENTIFICATION INFORMATION 1. Institution and Address Georgia Institute of Technology School of Physics Atlanta. Georgia 30332 2. NSF Program Biophysics Program 3. NSF Award Number PCM77-09726 4. Award Period From3/1/76 	To5/31/80 5. Cumulative Award Amount $71,000 6. Project Title 	 • The Transmission of Conformational Stability thr(Igh Adjacent Regions of DNA PART II—SUMMARY OF COMPLETED PROJECT (FOR PUBLIC USE) 	 • The dynamic and cooperative properties of DNA can be expected to play a role in the binding of gene regulatory proteins to specific DNA sites. 	It is the purpose of this project to further,understanding of the nature, range, and magnitude of the in- fluence of one DNA region on adjacent ones. 	Two approaches were employed. 	One approach was to examine the base pair opening transition of DNA molecules of known base pair sequences. 	Eperimental DNA denaturation transitions were compared to theoretical 	. predictions of the process for both long and short DNA molecules. 	The theory success- fully predicted the denaturation transitions of several DNAs with simple repeating base pair sequences as well as natural DNAs with more complex sequences. 	The theory was then used to examine the affect a protein binding to a specific DNA site has on adjacent regions. 	It was found that the magnitude of the affect depends on both the base pair sequence of the protein site as well as the adjacent xegions. 	At 37 ° C, the dnfluence is generally weak and of short range. 	A second approach employed Raman spectroscopy to examine the vibrational motion of differentgroups of DNA..atoms.....A method vas. 	. developed which enables one .to follow DNA vibrational bands during the 'B' to 'A' con- formational change. 	This change in DNA conformation may occurduring transcription initiation. 	Changes in certain backbone vibrations initiate the B to A transition whereas the transition curves of other vibrations undergo rapid and coupled changes. The results provide information on the conformational. pathway of•the B to A transition. . 	 - PART III—TECHNICAL INFORMATION (FOR PROGRAM MANAGEMENT USES) • 	 • ITEM (Check appropriate blocks) NONE ATTACHED V 	 )111# PREVIOUSLY FURNISHED TO BE FURNISHED SEPARATELY TO PROGRAM Check (/) • Approx. Date a. Abstracts of Theses b. Publication Citations c. Data on Scientific Collaborators Llf d. Information on Inventions a. 	Technical Description of Project and Results ,v>.;".:P., ,,I•:\,::,, ,":,,:s7 ,, ,',-':, A\\ - . '''.  ' . ."\k,,• ,:, 	:, ,:',• . : ,̀ . 1 l'f. 	Other (specify) 2. Principal Investigator/Project Director Name (Typed) Roger M. Wartell 	• ... wr 	f ,,,t,,,e;‘,..,.. m  3. Principal Investigator/Pro 	r o . 4. Date 	• 41, eS 1980 & 	' 	• . 	 us tOIt,ons 	 hnm Apsiso‘c4 11\111 \o IIIa. Abstract of PhD. thesis of Adrian Oliver, School of Physics, Georgia Tech Abstract The helix-coil transitions of five synthetic DNA polymers were experi-mentally examined. These periodic DNA polymers were d(A) n •d(T) n, d(AT) n •d(AT) n , d(AAT) n •d(ATT) n , d(G) II •d(C) n and d(GC) n •d(GC) n . The temperature induced transitions were monitored by ultraviolet absorbance. Transition curves for the A•T DNAs were analyzed theoretically'using the loop entropy model of DNA transi-tions. These DNA polymers allowed for a test of the accuracy of the DNA transi-tion theory and evaluation of DNA stability parameters. Results show excellent - overall agreement between theory and experiment. A loop entropy exponent R = 1.55 + .05 provided a best fit for all polymer transitions. • III b. Publication Citations  Published  1. "Helix-Coil Transition of d(A)n•d(T)n, d(AT) nod(AT) n and d(AAT) nild(ATT) n ; Evaluation of Stability Parameter of DNA," Biopolymers 16, 1115, 1977 by A. L. Oliver, R. M. Wartell and R. Ratliff. 2. "The Transmission of Stability or Instability from Site Specific protein-DNA Complexes," Nucleic Acid Research 4, 2779, 1977 by R. M. Wartell. 3. "Transition of Two Stranded DNA to Single Strands," in Statistical Mechanics  and Statistical Methods in Theory and Application  by R. M. Wartell, ed. U. Landman, p. 613, Plenum Pub., 1977. 4. "Minimum Energy Conformations of DNA Dimeric Subunits," Biopolymers 17, 1485, 1978 by S. Broyde, R. M. Wartell, S. D. Stellman and B. Hirgerty. 5. "Cloning DNA Restriction Endonuclease Fragments with Protruding Single Stranded Ends," Gene 9, 307, 1980 by R. M. Wartell and W. S. Reznikoff. Planned or Submitted  6. "Base Pair Opening in Promoter Sequences at Temperatures Below the Helix-Coil Transition," (Submitted) by R. M. Wartell and A. S. Benight. 7. "Agreement of Theory with Experimental Thermal Denaturation of Short DNA Restriction Fragments Containing Lactose Operator-Ptomoter Sequence." (Submitted) by A. S. Benight, R. M. Wartell and D. K. Howell. 8. "The B to A Transition of Calf Thymus DNA; Changes in Ten Raman Vibrational Bands During the Transition," by J. C. Martin and R. M. Wartell (in preparation). 9. "The Helix-Coil Transitions of d(G) n .d(C) n and d(I) 11. d(C) n ; Factors Effecting Reversibility and the Evaluation of Thermodynamic Parameters" by D. K. Howell and R. M. Wartell (planned). 10. "Experimental and Theoretical Thermal Denaturation Studies on Small Overlapping Restriction Fragments Containing the Escherichia cola  Lactose Genetic Control Region," W. Hillen, T. Goodman, S. Benight, R. M. Wartcal and R. D. Wells (in preparation). III c. 	Scientific Collaborators Status During Project Period of Association Collaborator 1. Dr. Adrian L. Oliver Graduate Student 1976-77 2. Dr. Rino Salvo Research Associate 1976-79 3. Dr. James Martin Postdoctoral Associate 1978 4. Mr. Dennis K. Howell Graduate Student 1977-present 5. Mr. Albert S. Benight Graduate Student 1979-present Collaborators at Other Institutions 6. Prof. William Reznikoff Professor (Univ. of Wisc.) Aug. 1978 - June 1979 7. Prof. Robert D. Wells Professor (Univ. of Wisc.) 1979-80 III e. Technical Summary  Experimental and theoretical methods have been employed to examine conformational changes transmitted through DNA. Emphasis has been placed on thermally induced base pair opening in duplex DNA, and the conformation of DNA as indicated by laser Raman spectroscopy. The thermally induced helix-coil transition of long synthetic DNA polymers, and short (80-300 base pairs) DNA restriction fragments of known sequence have been examined. The objective is to compare the DNA helix-coil transition theory with experiment, and"develope a simple predictive theory of base pair opening. Absorbance spectroscopy using computerized data collection and analysis was used to monitor the transitions of three A-T-polymers, poly d(A)• poly d(T), poly d(AT)• poly d(AT) and poly d(AAT)•poly d(ATT) and poly d(I)• poly d(C) and poly d(G)•poly d(C). Solvent conditions were found which made transitions of all DNA polymers reversible. This is required in order to confidently apply an equilibrium analysis. Although this was not a problem for the A•T polymers or poly d(I)•poly d(C), poly d(G)•poly d(C) does not give a reversible transition under commonly used solvent conditions (pH = 7, 0.001 M + 0.1 M NaCl). Poly d(G)•polyd(C) was found to be reversible in 0.05 M Na+, pH = 9.5. Analysis of reversible transitions gave similar values for the loop entropy factor for all DNAs. Stacking energies of adjacent base pairs were also evaluated.1 The helix-coil transition theory was also compared to experimental curves of four endonuclease restriction fragments of the transcription initiation region of the lactose operon. After appropriate modification of the theory to account for' strand dissociation, excellent agreement was obtained (Benight et al., enclosed preprint).2 The theory was used to calculate the transmission of stability and instability along DNA at temperatures below the helix coil transition region. Results show that around 37 °C at 0.2 M sodium solution the opening of base pairs at one site has a very small effect on the stability of sites 20 or more base pairs away. However, the transmitted affect increases rapidly with temperature and is influenced by the base sequence of the opened region and the immediately adjacent region. 3 It was desired to carry out experimental studies on short DNAs containing specific binding sites for RNA polymerase. Two restriction fragments of the lactose operon transcription initiation region were cloned.l+ The cloning procedure developed was a general one enabling the subsequent isolation of the cloned fragment. One of the fragments is 144 bps and is the shortest isolated DNA containing the binding sites for the catabolite activator protein, RNA polymerase and the lac repressor. 0.5 mgs of fragment has been isolated and is currently being studied. A new method has been developed which allows one to examine the B to A conformational transition of DNA by Raman spectroscopy. 5 The intensities of 10 DNA vibrational bands have been followed as a function of ethanol concen-tration [60 - 85% ethanol/water (vol/vol)]. The midpoint of the transition occurs at 72% ethanol. The transition curves of some of the backbone vibrations and base vibrations are similar, indicating a coupling between the conformational changes affecting these vibrations. Some Raman bands show interesting transition shapes. In particular, a unique pretransition change occurs for a deoxy ribose-phosphate vibration. This indicates that a change in conformation involving the deoxy ribose and/or phosphate group preceeds the onset of the B to A conform-ational change. It is planned to apply this Raman method to DNA molecules with known sequences. References  1. a) A. Oliver, R. M. Wartell and R. Ratliff, Biopolymers 16, 1115 (1977). b) D. K. Howell and R. M. Wartell, unpublished results. 2. a) A. S. Benight, R. M. Wartell and D. K. Howell, unpublished results. b) W. Hillen, T. Goodman, A. S. Benight, R. M. Wartell and R. D. Wells, unpublished results. 3. a) R. M. Wartell, Nuc. Acids Research 4, 2779 (1977). b) R. M. Wartell and A. S. Benight, unpublished results. 4. R. M. Wartell and W. S. Reznikoff, Gene 9, 307 (1980). 5. 1 J. C. Martin and R. M. Wartell, unpublished results. 

Transmission of conformational stability through adjacent regions of DNA

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Transmission of conformational stability through adjacent regions of DNA

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