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Phage Strategies Timothy G. Standish, Ph. D.
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The l Regulatory Region
Terminator left N utilization left N utilization right Terminator right nutR tL tR1 nutL cI cro cII cIII N Positive regulator Anti-terminator Repressor Anti-repressor Positive regulator “c” = clear referring to the clear plaques resulting from functional c genes
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The l Regulatory Region
Promoter Represser Establish-ment Promoter/ Operator left Promoter Represser Maintainence Promoter/ Operator right OR/PR PL/OL PRM PRE nutR tL tR1 nutL cI cro cII cIII N Positive regulator Anti-terminator Repressor Anti-repressor Positive regulator “c” = clear referring to the clear plaques resulting from functional c genes
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The l Regulatory Region: Infection
Host RNA Polymerase recognizes PL and PR and begins transcription of the N and cro proteins PL/OL PRM OR/PR PRE tL tR1 nutL nutR cI cro cII cIII N STOP Transcription Transcription
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The l Regulatory Region: Infection - Establishment of Lysogeny
Host RNA Polymerase recognizes PL and PR and begins transcription of the N and cro proteins PL/OL PRM OR/PR PRE tL tR1 nutL nutR cI cro cII cIII N As they are the very first genes to be expressed, N and cro are called “immediate early” genes. cro pN
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The l Regulatory Region: Infection - Establishment of Lysogeny
pN binds to nut sites allowing transcription through tR1 and tL PL/OL PRM OR/PR PRE tL tR1 nutL nutR cI cro cII cIII N cro pN
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The l Regulatory Region: Infection - Establishment of Lysogeny
pN binds to nut sites allowing transcription through tR1 and tL PL/OL PRm OR/PR PRE tL tR1 nutL nutR cI cro cII cIII N pN pN Transcription cro
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The l Regulatory Region: Infection - Establishment of Lysogeny
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. PL/OL PRm OR/PR PRE tL tR1 nutL nutR cI cro cII cIII N cII cIII pN pN cII cIII cro
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The l Regulatory Region: Infection - Establishment of Lysogeny
Transcription from PRE makes mRNA complementary to the cro mRNA thus inhibiting cro translation PL/OL PRm OR/PR PRE tL tR1 nutL nutR cI cro cII cIII N cII cIII pN pN Transcription cro
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The l Regulatory Region: Infection - Establishment of Lysogeny
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. PL/OL PRm OR/PR PRE tL tR1 nutL nutR cI cro cII cIII N cII cIII pN pN Transcription
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The l Regulatory Region: Infection - Establishment of Lysogeny
The repressor gene product of cI binds to OR and OL preventing transcription from PR and PL. PL/OL PRm OR/PR PRE tL tR1 nutL nutR cI cro cII cIII N cII cIII pN cI pN cI
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The l Regulatory Region: Infection - Establishment of Lysogeny
The repressor up-regulates expression from PRM maintaining repressor production and preventing expression of any other proteins and establishing lysogeny. PL/OL PRM OR/PR PRE tL tR1 nutL nutR cI cro cII cIII N cI cI Transcription
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The l Repressor Protein
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 A 40 residue connector runs Between the two domains C N Repressor C N Repressor dimer Repressor only binds DNA efficiently when it is a dimer
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The l Regulatory Region: Infection - Establishment of The Lytic Cycle
Host RNA Polymerase recognizes PL and PR and begins transcription of the N and cro proteins PL/OL PRM OR/PR PRE tL tR1 nutL nutR cI cro cII cIII N As they are the very first genes to be expressed, N and cro are called “immediate early” genes. cro pN
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The l Regulatory Region: Infection - Establishment of The Lytic Cycle
Host RNA Polymerase recognizes PL and PR and begins transcription of the N and cro proteins OR/PR cI cro PRM
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The l Regulatory Region: Infection - Establishment of The Lytic Cycle
Host RNA Polymerase recognizes PL and PR and begins transcription of the N and cro proteins OR/PR 5’CAT’ACGTTAAATCTATCACCGCAAGGGATAAATATCTAACACCGTGCGTGTTGACTATTTTACCTCTGGCGGTGATAATGGTTGC’ATG3’ 3’ATG’TGCAATTTAGATAGTGGCGTTCCCTATTTATAGATTGTGGCACGCACAACTGATAAAATGGAGACCGCCACTATTACCAACG’TAC5’ cI PRM OR2 OR1 OR3 cro
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The l Regulatory Region: Infection - Establishment of The Lytic Cycle
5’CAT’ACGTTAAATCTATCACCGCAAGGGATAAATATCTAACACCGTGCGTGTTGACTATTTTACCTCTGGCGGTGATAATGGTTGC’ATG3’ 3’ATG’TGCAATTTAGATAGTGGCGTTCCCTATTTATAGATTGTGGCACGCACAACTGATAAAATGGAGACCGCCACTATTACCAACG’TAC5’ cI OR2 OR1 OR3 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
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The l Regulatory Region: Infection - Establishment of The Lytic Cycle
Host RNA Polymerase recognizes PL and PR and begins transcription of the N and cro proteins RNA Polymerase 5’CAT’ACGTTAAATCTATCACCGCAAGGGATAAATATCTAACACCGTGCGTGTTGACTATTTTACCTCTGGCGGTGATAATGGTTGC’ATG3’ 3’ATG’TGCAATTTAGATAGTGGCGTTCCCTATTTATAGATTGTGGCACGCACAACTGATAAAATGGAGACCGCCACTATTACCAACG’TAC5’ RNA Polymerase cI OR2 OR1 OR3 cro
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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.
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The End
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