1. ©2001 Timothy G. Standish Exodus 15:26 26… If thou wilt diligently hearken to the voce of the LORD thy God, and wilt do that which is right in his sight, and wilt give ear to his commandments, and keep all his statutes, I will put none of these diseases upon thee, which I have brought upon the Egyptians: for I am the LORD that healeth thee.

2. ©2001 Timothy G. Standish Phage Strategies Timothy G. Standish, Ph. D.

3. ©2001 Timothy G. Standish

4. Phage Reproduction: The Lytic Cycle Destruction of the bacteria’s DNA Replication of the viral genome Production of viral parts Packaging Infection Lysis

5. ©2001 Timothy G. Standish Many generations of bacteria Temperate phage Phage Reproduction: The Lysogenic Cycle Integration of phage DNA into the bacterial genome Circularization of phage DNA Infection On to the lytic cycle Exit of phage

6. ©2001 Timothy G. Standish The Genome cos Head Genes Tail Genes Recombination Lysis Replication Regulation CI cro CII O Z U  int xis att N CIII   P Q C Nu3 D E F II S R A W B FIFI L K I J V G T H M tLtL nut L t R3 t RI nut R P L /O L P RM P RE P R /O R PRPR

7. ©2001 Timothy G. Standish cos Head Genes Tail Genes Recombination Lysis Replication Regulation CI cro CII O Z U  int xis att N CIII   P Q C Nu3 D E F II S R A W B FIFI L K I J V G T H M Late Order of Gene Expression Immediate Early Immediate Early Delayed Early Delayed Early

8. ©2001 Timothy G. Standish The Regulatory Region cI cro cII cIII N N nut R tLtL t R1 nut L RepressorPositive regulator Anti- repressor Anti- terminator Positive regulator N utilization right Terminator right N utilization left Terminator left “c” = clear referring to the clear plaques resulting from functional c genes

9. ©2001 Timothy G. Standish The Regulatory Region cI cro cII cIII N N RepressorPositive regulator Anti- repressor Anti- terminator Positive regulator Promoter Repressor Establish- ment Promoter/ Operator right Promoter/ Operator left Promoter Repressor Maintainence “c” = clear referring to the clear plaques resulting from functional c genes O R /P R P L /O L P RM P RE nut R tLtL t R1 nut L

10. ©2001 Timothy G. Standish STOP Transcription STOP The Regulatory Region: Infection cI cro cII cIII N N nut R tLtL t R1 O R /P R P L /O L nut L P RM Host RNA Polymerase recognizes P L and P R and begins transcription of the N and cro proteins Transcription P RE

11. ©2001 Timothy G. Standish The Regulatory Region: Infection - Establishment of Lysogeny cI cro cII cIII N N nut R tLtL t R1 O R /P R P L /O L nut L P RE P RM cro Host RNA Polymerase recognizes P L and P R and begins transcription of the N and cro proteins pN As they are the very first genes to be expressed, N and cro are called “immediate early” genes.

12. ©2001 Timothy G. Standish The Regulatory Region: Infection - Establishment of Lysogeny cI cro cII cIII N N nut R tLtL t R1 O R /P R P L /O L nut L P RE P RM pN binds to nut sites allowing transcription through t R1 and t L cro pN

13. ©2001 Timothy G. Standish The Regulatory Region: Infection - Establishment of Lysogeny cI cro cII cIII N N nut R tLtL t R1 O R /P R P L /O L nut L P RE P Rm pN binds to nut sites allowing transcription through t R1 and t L cro pN Transcription

14. ©2001 Timothy G. Standish The Regulatory Region: Infection - Establishment of Lysogeny cI cro cII cIII N N nut R tLtL t R1 O R /P R P L /O L nut L P RE P Rm The cII and cIII proteins are produced. In vivo a protein called HflA rapidly breaks down cII unless it is combined with cIII. cII up regulates transcription from P RE. cro pN cII cIII cII cIII

15. ©2001 Timothy G. Standish The Regulatory Region: Infection - Establishment of Lysogeny cI cro cII cIII N N nut R tLtL t R1 O R /P R P L /O L nut L P RE P Rm Transcription from P RE makes mRNA complimentary to the cro mRNA thus inhibiting cro translation cro pN cII cIII Transcription

16. ©2001 Timothy G. Standish The Regulatory Region: Infection - Establishment of Lysogeny cI cro cII cIII N N nut R tLtL t R1 O R /P R P L /O L nut L P RE P Rm The transcript from PRE provides a 3’ untranslated region with an efficient ribosome binding site before the start of the cI gene, thus cI repressor is efficiently produced. pN cII cIII Transcription

17. ©2001 Timothy G. Standish The Regulatory Region: Infection - Establishment of Lysogeny cI cro cII cIII N N nut R tLtL t R1 O R /P R P L /O L nut L P RE P Rm The repressor gene product of cI binds to O R and O L preventing transcription from P R and P L. pN cII cIII cI

18. ©2001 Timothy G. Standish The Regulatory Region: Infection - Establishment of Lysogeny cI cro cII cIII N N nut R tLtL t R1 O R /P R P L /O L nut L P RE P RM The repressor up-regulates expression from P RM maintaining repressor production and preventing expression of any other proteins and establishing lysogeny. cI Transcription

19. ©2001 Timothy G. Standish The Repressor Protein Repressor protein has two functional domains: 1 An amino acid (residue) 1-92 N-terminal DNA binding domain which binds to O R and O L 2 A C-terminal protein binding domain essential for dimerization running from amino acids 132-236 A 40 residue connector runs between the two domains C N Repressor C N C N Repressor dimer Repressor only binds DNA efficiently when it is a dimer

20. ©2001 Timothy G. Standish The Regulatory Region: Infection - Establishment of The Lytic Cycle cI cro cII cIII N N nut R tLtL t R1 O R /P R P L /O L nut L P RE P RM cro Host RNA Polymerase recognizes P L and P R and begins transcription of the N and cro proteins pN As they are the very first genes to be expressed, N and cro are called “immediate early” genes.

21. ©2001 Timothy G. Standish The Regulatory Region: Infection - Establishment of The Lytic Cycle cI cro O R /P R P RM Host RNA Polymerase recognizes P L and P R and begins transcription of the N and cro proteins

22. ©2001 Timothy G. Standish The Regulatory Region: Infection - Establishment of The Lytic Cycle O R /P R P RM Host RNA Polymerase recognizes P L and P R and begins transcription of the N and cro proteins cI cro 5’ CAT’ACGTTAAATCTATCACCGCAAGGGATAAATATCTAACACCGTGCGTGTTGACTATTTTACCTCTGGCGGTGATAAT GGTTGC’ATG 3’ 3’ ATG’TGCAATTTAGATAGTGGCGTTCCCTATTTATAGATTGTGGCACGCACAACTGATAAAATGGAGACCGCCACTATTA CCAACG’TAC 5’ 5’ CAT’ACGTTAAATCTATCACCGCAAGGGATAAATATCTAACACCGTGCGTGTTGACTATTTTACCTCTGGCGGTGATAAT GGTTGC’ATG 3’ 3’ ATG’TGCAATTTAGATAGTGGCGTTCCCTATTTATAGATTGTGGCACGCACAACTGATAAAATGGAGACCGCCACTATTA CCAACG’TAC 5’ OR2OR2 OR2OR2 OR1OR1 OR1OR1 OR3OR3 OR3OR3

23. ©2001 Timothy G. Standish The N terminus of the repressor binds to O R 1 and 2 with high affinity cI cro 5’ CAT’ACGTTAAATCTATCACCGCAAGGGATAAATATCTAACACCGTGCGTGTTGACTATTTTACCTCTGGCGGTGATAAT GGTTGC’ATG 3’ 3’ ATG’TGCAATTTAGATAGTGGCGTTCCCTATTTATAGATTGTGGCACGCACAACTGATAAAATGGAGACCGCCACTATTA CCAACG’TAC 5’ 5’ CAT’ACGTTAAATCTATCACCGCAAGGGATAAATATCTAACACCGTGCGTGTTGACTATTTTACCTCTGGCGGTGATAAT GGTTGC’ATG 3’ 3’ ATG’TGCAATTTAGATAGTGGCGTTCCCTATTTATAGATTGTGGCACGCACAACTGATAAAATGGAGACCGCCACTATTA CCAACG’TAC 5’ OR2OR2 OR2OR2 OR1OR1 OR1OR1 OR3OR3 OR3OR3 C N C N C N The Regulatory Region: Infection - Establishment of The Lytic Cycle The N terminus of the repressor binds to O R 1 and 2 with high affinity

24. ©2001 Timothy G. Standish RNA Polymerase RNA Polymerase RNA Polymerase RNA Polymerase The Regulatory Region: Infection - Establishment of The Lytic Cycle Host RNA Polymerase recognizes P L and P R and begins transcription of the N and cro proteins cI cro 5’ CAT’ACGTTAAATCTATCACCGCAAGGGATAAATATCTAACACCGTGCGTGTTGACTATTTTACCTCTGGCGGTGATAAT GGTTGC’ATG 3’ 3’ ATG’TGCAATTTAGATAGTGGCGTTCCCTATTTATAGATTGTGGCACGCACAACTGATAAAATGGAGACCGCCACTATTA CCAACG’TAC 5’ 5’ CAT’ACGTTAAATCTATCACCGCAAGGGATAAATATCTAACACCGTGCGTGTTGACTATTTTACCTCTGGCGGTGATAAT GGTTGC’ATG 3’ 3’ ATG’TGCAATTTAGATAGTGGCGTTCCCTATTTATAGATTGTGGCACGCACAACTGATAAAATGGAGACCGCCACTATTA CCAACG’TAC 5’ OR2OR2 OR2OR2 OR1OR1 OR1OR1 OR3OR3 OR3OR3

25. ©2001 Timothy G. Standish The Regulatory Region: Infection - Establishment of The Lytic Cycle Right and left operator sites share a common consensus sequence. O R 3 CTATCACCGCAAGGGA TAA O R 2 CTAACACCGTGCGTGT TGA O R 1 TTACCTCTGGCGGTGA TAA O L 3 TAACCATCTGCGGTGA TAA O L 2 TTATCTCTGGCGGTGTT GA O L 1 ATACCACTGGCGGTGA TAC O R 3 CTATCACCGCAAGGGA TAA O R 2 CTAACACCGTGCGTGT TGA O R 1 TTACCTCTGGCGGTGA TAA O L 3 TAACCATCTGCGGTGA TAA O L 2 TTATCTCTGGCGGTGTT GA O L 1 ATACCACTGGCGGTGA TAC CON- TAYCWCYGGCGGTGWTR-

26. ©2001 Timothy G. Standish