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.


Phage Phage StrategiesStrategies

Timothy G. Standish, Ph. D.



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


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


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


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


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


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


STOPTranscriptionTranscription

STOP

The The Regulatory Region: Regulatory Region:InfectionInfection


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


The The Regulatory Region: Regulatory Region:Infection - Establishment of LysogenyInfection - Establishment of Lysogeny


nutRtL tR1

OR/PRPL/OL

nutL

PREPRM

crocro


pN

As they are the very first genes to be expressed, N and cro are called “immediate early” genes.




nutRtL tR1

OR/PRPL/OL

nutL

PREPRM

pN binds to nut sites allowing transcription through tR1 and tL

crocropN




nutRtL tR1

OR/PRPL/OL

nutL

PREPRm

pN binds to nut sites allowing transcription through tR1 and tL

crocro

pN pN

TranscriptionTranscription




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




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




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




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




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


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


The The Regulatory Region: Regulatory Region:Infection - Establishment of The Lytic CycleInfection - Establishment of The Lytic Cycle


nutRtL tR1

OR/PRPL/OL

nutL

PREPRM

crocro


pN

As they are the very first genes to be expressed, N and cro are called “immediate early” genes.



cIcI crocroOR/PR

PRM




OR/PR

PRM


cIcI crocro

5’CAT’ACGTTAAATCTATCACCGCAAGGGATAAATATCTAACACCGTGCGTGTTGACTATTTTACCTCTGGCGGTGATAATGGTTGC’ATG3’

3’ATG’TGCAATTTAGATAGTGGCGTTCCCTATTTATAGATTGTGGCACGCACAACTGATAAAATGGAGACCGCCACTATTACCAACG’TAC5’


3’ATG’TGCAATTTAGATAGTGGCGTTCCCTATTTATAGATTGTGGCACGCACAACTGATAAAATGGAGACCGCCACTATTACCAACG’TAC5’OR2OR2 OR1OR1OR3OR3


The N terminus of the repressor binds to OR1 and 2 with high affinity

cIcI crocro





C

N

C

N

C

N





RNA PolymeraseRNA Polymerase

RNA Polymerase

RNA Polymerase



cIcI crocro







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-


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Exodus 15:26