Exodus 15:26
description
Transcript of Exodus 15:26
©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.
©2001 Timothy G. Standish
Phage Phage StrategiesStrategies
Timothy G. Standish, Ph. D.
©2001 Timothy G. Standish
Phage Reproduction:Phage Reproduction:The Lytic CycleThe Lytic Cycle
Destruction of the bacteria’s DNA
Replication of the viral genome
Production of viral parts
Packaging
Infection
Lysis
©2001 Timothy G. Standish
Many generations of bacteria
Temperate phage
Phage Reproduction:Phage Reproduction:The Lysogenic CycleThe Lysogenic Cycle
Integration of phage DNA into the bacterial genome
Circularization of phage DNA
Infection
On to the lytic cycle
Exit of phage
©2001 Timothy G. Standish
The The Genome Genome
cos
Head GenesHead Genes
Tail GenesTail Genes
RecombinationRecombination LysisLysis
ReplicationReplication
RegulationRegulation
CI croCII
O
ZU
int
xis
att
NCIII
PQ
CNu3D
E
FII
SR
AWB
FI
LKIJ
VGTHM
tLnutL
tR3
tRInutR P L/O L
PRMPREPR/OR
PR
©2001 Timothy G. Standish
cos
Head GenesHead Genes
Tail GenesTail Genes
RecombinationRecombination LysisLysis
ReplicationReplication
RegulationRegulation
CI croCII
O
ZU
int
xis
att
NCIII
PQ
CNu3D
E
FII
SR
AWB
FI
LKIJ
VGTHM
LateLate
Order of Gene Order of Gene ExpressionExpression
Immediate EarlyImmediate Early
DelayedEarly
DelayedEarly
©2001 Timothy G. Standish
The The Regulatory Region Regulatory Region
cIcI crocro cIIcIIcIIIcIII NN
nutRtL tR1nutL
Repressor Positive regulator
Anti-repressor
Anti-terminator
Positive regulator
N utilizationright
Terminatorright
N utilizationleft
Terminatorleft
“c” = clear referring to the clear plaques resulting from functional c genes
©2001 Timothy G. Standish
The The Regulatory Region Regulatory Region
cIcI crocro cIIcIIcIIIcIII NNRepressor Positive
regulatorAnti-repressor
Anti-terminator
Positive regulator
Promoter Represser Establish-ment
Promoter/ Operator right
Promoter/ Operator left
Promoter Represser Maintainence
“c” = clear referring to the clear plaques resulting from functional c genes
OR/PRPL/OL PRM PRE
nutRtL tR1nutL
©2001 Timothy G. Standish
STOPTranscriptionTranscription
STOP
The The Regulatory Region: Regulatory Region:InfectionInfection
cIcI crocro cIIcIIcIIIcIII NN
nutRtL tR1
OR/PRPL/OL
nutL
PRM
Host RNA Polymerase recognizes PL and PR and begins transcription of the N and cro proteins
TranscriptionTranscription
PRE
©2001 Timothy G. Standish
The The Regulatory Region: Regulatory Region:Infection - Establishment of LysogenyInfection - Establishment of Lysogeny
cIcI crocro cIIcIIcIIIcIII NN
nutRtL tR1
OR/PRPL/OL
nutL
PREPRM
crocro
Host RNA Polymerase recognizes PL and PR 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.
©2001 Timothy G. Standish
The The Regulatory Region: Regulatory Region:Infection - Establishment of LysogenyInfection - Establishment of Lysogeny
cIcI crocro cIIcIIcIIIcIII NN
nutRtL tR1
OR/PRPL/OL
nutL
PREPRM
pN binds to nut sites allowing transcription through tR1 and tL
crocropN
©2001 Timothy G. Standish
The The Regulatory Region: Regulatory Region:Infection - Establishment of LysogenyInfection - Establishment of Lysogeny
cIcI crocro cIIcIIcIIIcIII NN
nutRtL tR1
OR/PRPL/OL
nutL
PREPRm
pN binds to nut sites allowing transcription through tR1 and tL
crocro
pN pN
TranscriptionTranscription
©2001 Timothy G. Standish
The The Regulatory Region: Regulatory Region:Infection - Establishment of LysogenyInfection - Establishment of Lysogeny
cIcI crocro cIIcIIcIIIcIII NN
nutRtL tR1
OR/PRPL/OL
nutL
PREPRm
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.
crocro
pN pN cIIcIII
cIIcIII
©2001 Timothy G. Standish
The The Regulatory Region: Regulatory Region:Infection - Establishment of LysogenyInfection - Establishment of Lysogeny
cIcI crocro cIIcIIcIIIcIII NN
nutRtL tR1
OR/PRPL/OL
nutL
PREPRm
Transcription from PRE makes mRNA complimentary to the cro mRNA thus inhibiting
cro translation
crocro
pN pN cIIcIII
Transcription
©2001 Timothy G. Standish
The The Regulatory Region: Regulatory Region:Infection - Establishment of LysogenyInfection - Establishment of Lysogeny
cIcI crocro cIIcIIcIIIcIII NN
nutRtL tR1
OR/PRPL/OL
nutL
PREPRm
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 pN cIIcIII
Transcription
©2001 Timothy G. Standish
The The Regulatory Region: Regulatory Region:Infection - Establishment of LysogenyInfection - Establishment of Lysogeny
cIcI crocro cIIcIIcIIIcIII NN
nutRtL tR1
OR/PRPL/OL
nutL
PREPRm
The repressor gene product of cI binds to OR and OL preventing transcription from PR and PL.
pN pN cIIcIII
cIcI
cIcI cIcI
©2001 Timothy G. Standish
The The Regulatory Region: Regulatory Region:Infection - Establishment of LysogenyInfection - Establishment of Lysogeny
cIcI crocro cIIcIIcIIIcIII NN
nutRtL tR1
OR/PRPL/OL
nutL
PREPRM
The repressor up-regulates expression from PRM maintaining repressor production and preventing
expression of any other proteins and establishing lysogeny.
cIcI cIcI
Transcription
©2001 Timothy G. Standish
The The Repressor Protein Repressor Protein Repressor protein has two functional domains:
1 An amino acid (residue) 1-92 N-terminal DNA binding domain which binds to OR and OL
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
©2001 Timothy G. Standish
The The Regulatory Region: Regulatory Region:Infection - Establishment of The Lytic CycleInfection - Establishment of The Lytic Cycle
cIcI crocro cIIcIIcIIIcIII NN
nutRtL tR1
OR/PRPL/OL
nutL
PREPRM
crocro
Host RNA Polymerase recognizes PL and PR 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.
©2001 Timothy G. Standish
The The Regulatory Region: Regulatory Region:Infection - Establishment of The Lytic CycleInfection - Establishment of The Lytic Cycle
cIcI crocroOR/PR
PRM
Host RNA Polymerase recognizes PL and PR and begins transcription of the N and cro proteins
©2001 Timothy G. Standish
The The Regulatory Region: Regulatory Region:Infection - Establishment of The Lytic CycleInfection - Establishment of The Lytic Cycle
OR/PR
PRM
Host RNA Polymerase recognizes PL and PR and begins transcription of the N and cro proteins
cIcI crocro
5’CAT’ACGTTAAATCTATCACCGCAAGGGATAAATATCTAACACCGTGCGTGTTGACTATTTTACCTCTGGCGGTGATAATGGTTGC’ATG3’
3’ATG’TGCAATTTAGATAGTGGCGTTCCCTATTTATAGATTGTGGCACGCACAACTGATAAAATGGAGACCGCCACTATTACCAACG’TAC5’
5’CAT’ACGTTAAATCTATCACCGCAAGGGATAAATATCTAACACCGTGCGTGTTGACTATTTTACCTCTGGCGGTGATAATGGTTGC’ATG3’
3’ATG’TGCAATTTAGATAGTGGCGTTCCCTATTTATAGATTGTGGCACGCACAACTGATAAAATGGAGACCGCCACTATTACCAACG’TAC5’OR2OR2 OR1OR1OR3OR3
©2001 Timothy G. Standish
The N terminus of the repressor binds to OR1 and 2 with high affinity
cIcI crocro
5’CAT’ACGTTAAATCTATCACCGCAAGGGATAAATATCTAACACCGTGCGTGTTGACTATTTTACCTCTGGCGGTGATAATGGTTGC’ATG3’
3’ATG’TGCAATTTAGATAGTGGCGTTCCCTATTTATAGATTGTGGCACGCACAACTGATAAAATGGAGACCGCCACTATTACCAACG’TAC5’
5’CAT’ACGTTAAATCTATCACCGCAAGGGATAAATATCTAACACCGTGCGTGTTGACTATTTTACCTCTGGCGGTGATAATGGTTGC’ATG3’
3’ATG’TGCAATTTAGATAGTGGCGTTCCCTATTTATAGATTGTGGCACGCACAACTGATAAAATGGAGACCGCCACTATTACCAACG’TAC5’OR2OR2 OR1OR1OR3OR3
C
N
C
N
C
N
The The Regulatory Region: Regulatory Region:Infection - Establishment of The Lytic CycleInfection - Establishment of The Lytic Cycle
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
©2001 Timothy G. Standish
RNA PolymeraseRNA Polymerase
RNA Polymerase
RNA Polymerase
The The Regulatory Region: Regulatory Region:Infection - Establishment of The Lytic CycleInfection - Establishment of The Lytic Cycle
Host RNA Polymerase recognizes PL and PR and begins transcription of the N and cro proteins
cIcI crocro
5’CAT’ACGTTAAATCTATCACCGCAAGGGATAAATATCTAACACCGTGCGTGTTGACTATTTTACCTCTGGCGGTGATAATGGTTGC’ATG3’
3’ATG’TGCAATTTAGATAGTGGCGTTCCCTATTTATAGATTGTGGCACGCACAACTGATAAAATGGAGACCGCCACTATTACCAACG’TAC5’
5’CAT’ACGTTAAATCTATCACCGCAAGGGATAAATATCTAACACCGTGCGTGTTGACTATTTTACCTCTGGCGGTGATAATGGTTGC’ATG3’
3’ATG’TGCAATTTAGATAGTGGCGTTCCCTATTTATAGATTGTGGCACGCACAACTGATAAAATGGAGACCGCCACTATTACCAACG’TAC5’OR2OR2 OR1OR1OR3OR3
©2001 Timothy G. Standish
The The Regulatory Region: Regulatory Region:Infection - Establishment of The Lytic CycleInfection - Establishment of The Lytic Cycle
Right and left operator sites share a common consensus sequence.
OR3 CTATCACCGCAAGGGATAAOR2 CTAACACCGTGCGTGTTGAOR1 TTACCTCTGGCGGTGATAA
OL3 TAACCATCTGCGGTGATAAOL2 TTATCTCTGGCGGTGTTGAOL1 ATACCACTGGCGGTGATAC
OR3 CTATCACCGCAAGGGATAAOR2 CTAACACCGTGCGTGTTGAOR1 TTACCTCTGGCGGTGATAA
OL3 TAACCATCTGCGGTGATAAOL2 TTATCTCTGGCGGTGTTGAOL1 ATACCACTGGCGGTGATACCON-TAYCWCYGGCGGTGWTR-
©2001 Timothy G. Standish