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Phage Strategies

Phage Strategies. Timothy G. Standish, Ph. D. The l Regulatory Region. t R1. t L. nut L. nut R. N. cI. cro. cIII. cII. Terminator left. N utilization left. N utilization right. Terminator right. Positive regulator. Anti-terminator. Repressor. Anti-repressor.

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Phage Strategies

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  1. Phage Strategies Timothy G. Standish, Ph. D.

  2. The l Regulatory Region tR1 tL nutL nutR N cI cro cIII cII Terminator left N utilization left N utilization right Terminator right Positive regulator Anti-terminator Repressor Anti-repressor Positive regulator “c” = clear referring to the clear plaques resulting from functional c genes

  3. The l Regulatory Region PRM PRE PL/OL OR/PR tR1 tL nutL nutR N cI cro cIII cII Promoter Represser Establish-ment Promoter/ Operator left Promoter Represser Maintainence Promoter/ Operator right Positive regulator Anti-terminator Repressor Anti-repressor Positive regulator “c” = clear referring to the clear plaques resulting from functional c genes

  4. The l Regulatory Region:Infection PRM PRE PL/OL OR/PR tR1 tL nutL nutR N cI cro STOP STOP cIII cII Transcription Transcription Transcription Transcription Host RNA Polymerase recognizes PL and PR and begins transcription of the N and cro proteins

  5. The l Regulatory Region:Infection - Establishment of Lysogeny PRM PRE PL/OL OR/PR tR1 tL nutL nutR N cI cro cIII cII pN Host RNA Polymerase recognizes PL and PR and begins transcription of the N and cro proteins As they are the very first genes to be expressed, N and cro are called “immediate early” genes. cro

  6. The l Regulatory Region:Infection - Establishment of Lysogeny PRM PRE PL/OL OR/PR tR1 tL nutL nutR N cI cro cIII cII pN pN binds to nut sites allowing transcription through tR1 and tL cro

  7. The l Regulatory Region:Infection - Establishment of Lysogeny PRm PRE PL/OL OR/PR tR1 tL nutL nutR N cI cro pN pN cIII cII Transcription Transcription pN binds to nut sites allowing transcription through tR1 and tL cro

  8. The l Regulatory Region:Infection - Establishment of Lysogeny PRm PRE PL/OL OR/PR tR1 tL nutL nutR N cI cro pN pN cIII cII cII cII cIII cIII 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 PRE. cro

  9. The l Regulatory Region:Infection - Establishment of Lysogeny PRm PRE PL/OL OR/PR tR1 tL nutL nutR N cI cro pN pN cIII cII Transcription cII cIII Transcription from PRE makes mRNA complementary to the cro mRNA thus inhibiting cro translation cro

  10. The l Regulatory Region:Infection - Establishment of Lysogeny PRm PRE PL/OL OR/PR tR1 tL nutL nutR N cI cro pN pN cIII cII Transcription cII cIII 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.

  11. The l Regulatory Region:Infection - Establishment of Lysogeny PRm PRE PL/OL OR/PR tR1 tL nutL nutR N cI cro pN cI cI pN cIII cII cII cIII cI The repressor gene product of cI binds to OR and OL preventing transcription from PR and PL.

  12. The l Regulatory Region:Infection - Establishment of Lysogeny PRM PRE PL/OL OR/PR tR1 tL nutL nutR N cI cro cIII cII Transcription The repressor up-regulates expression from PRM maintaining repressor production and preventing expression of any other proteins and establishing lysogeny. cI cI

  13. The l Repressor Protein Repressor Repressor dimer C C C N N N • Repressor protein has two functional domains: • An amino acid (residue) 1-92 N-terminal DNA binding domain which binds to OR and OL • A C-terminal protein binding domain essential for dimerization running from amino acids 132-236 • A 40 residue connector runs Between the two domains Repressor only binds DNA efficiently when it is a dimer

  14. The l Regulatory Region:Infection - Establishment of The Lytic Cycle PRM PRE PL/OL OR/PR tR1 tL nutL nutR N cI cro cIII cII pN Host RNA Polymerase recognizes PL and PR and begins transcription of the N and cro proteins As they are the very first genes to be expressed, N and cro are called “immediate early” genes. cro

  15. The l Regulatory Region:Infection - Establishment of The Lytic Cycle OR/PR PRM Host RNA Polymerase recognizes PL and PR and begins transcription of the N and cro proteins cI cro

  16. The l Regulatory Region:Infection - Establishment of The Lytic Cycle OR/PR PRM OR3 OR2 OR1 Host RNA Polymerase recognizes PL and PR and begins transcription of the N and cro proteins 5’CAT’ACGTTAAATCTATCACCGCAAGGGATAAATATCTAACACCGTGCGTGTTGACTATTTTACCTCTGGCGGTGATAATGGTTGC’ATG3’ 3’ATG’TGCAATTTAGATAGTGGCGTTCCCTATTTATAGATTGTGGCACGCACAACTGATAAAATGGAGACCGCCACTATTACCAACG’TAC5’ cI cro

  17. C C C The l Regulatory Region:Infection - Establishment of The Lytic Cycle N N N OR3 OR2 OR1 5’CAT’ACGTTAAATCTATCACCGCAAGGGATAAATATCTAACACCGTGCGTGTTGACTATTTTACCTCTGGCGGTGATAATGGTTGC’ATG3’ 3’ATG’TGCAATTTAGATAGTGGCGTTCCCTATTTATAGATTGTGGCACGCACAACTGATAAAATGGAGACCGCCACTATTACCAACG’TAC5’ cI cro The N terminus of the repressor binds to OR1 and 2 with high affinity The N terminus of the repressor binds to OR1 and 2 with high affinity The N terminus of the repressor binds to OR1 and 2 with high affinity

  18. The l Regulatory Region:Infection - Establishment of The Lytic Cycle RNA Polymerase RNA Polymerase OR3 OR2 OR1 Host RNA Polymerase recognizes PL and PR and begins transcription of the N and cro proteins 5’CAT’ACGTTAAATCTATCACCGCAAGGGATAAATATCTAACACCGTGCGTGTTGACTATTTTACCTCTGGCGGTGATAATGGTTGC’ATG3’ 3’ATG’TGCAATTTAGATAGTGGCGTTCCCTATTTATAGATTGTGGCACGCACAACTGATAAAATGGAGACCGCCACTATTACCAACG’TAC5’ cI cro

  19. The l Regulatory Region:Infection - Establishment of The Lytic Cycle OR3 CTATCACCGCAAGGGATAA OR2 CTAACACCGTGCGTGTTGA OR1 TTACCTCTGGCGGTGATAA OL3 TAACCATCTGCGGTGATAA OL2 TTATCTCTGGCGGTGTTGA OL1 ATACCACTGGCGGTGATAC OR3 CTATCACCGCAAGGGATAA OR2 CTAACACCGTGCGTGTTGA OR1 TTACCTCTGGCGGTGATAA OL3 TAACCATCTGCGGTGATAA OL2 TTATCTCTGGCGGTGTTGA OL1 ATACCACTGGCGGTGATAC CON -TAYCWCYGGCGGTGWTR- Right and left operator sites share a common consensus sequence.

  20. The End

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