Phage Strategies Timothy G. Standish, Ph. D.

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

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

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

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

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

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

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

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

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

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

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

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 132-236 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

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

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

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

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

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

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.

The End