Transcript plaque a induction

Chapter 8
Major Shifts in
Bacterial Transcription
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Outline
•Sigma factors Switching
–Phage infection
–Sporulation
–Genes with multiple Promoters
–Other s switches
•The RNA polymerase Encoded in phage T7
•Infection of E.coli by phage 
–Lytic Reproduction of phage 
–Establishing Lysogeny
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
8.1 Sigma Factor Switching 轉換
•Phage infection of bacterium subverts host
transcription machinery 當噬菌體感染細菌時則會破壞
寄主本身的轉錄機制
–In process, establishes a time-dependent, or
temporal, program of transcription
•First early phage genes are transcribed
 Immediately early gene (即刻早期基因)
•This is followed by the later genes
 Early gene expression (早期基因)
•Late in the infectious cycle there is no longer
transcription of the host genes, only phage genes
 Late gene expression (晚期基因)
•Change in what genes transcribed is caused by a
change in transcription machinery, in RNA
polymerase itself
Phage Infection
• Chapter 6 established that s is the key factor
in determining specificity of T4 DNA
transcription
• To shift the transcription process s is a likely
candidate
• Study of the process done in B. subtilis 枯草
芽孢桿菌and its phage, SPO1
(NOT in E. coli system)
B. subtilis RNA polymerase
2a, b, b’, w, s (43kDa),
d (prevent binding to nonpromoter region)
s: 43kDa
Temporal Control of
Transcription
• Like T4, SPO1 has a large
DNA genome
• Temporal transcription
program:
– First 5 minutes:
expression of early genes
– During 5 – 10 minutes:
expression of middle
genes
– After 10 minutes to end:
late genes expressed
Material:
Phage SPO1-infected B. subtilis
change specificity
•Transcribe phage gene
•From early to middle
phage transcription
gp28: new s factor
Phage s factor
Host core polymerase
gp33, gp34: another s factor
Phage gp33, gp34
Host core polymerase
Transcription Switching
• This switching is directed by a set of phageencoded s factors that associate with
the host core RNA polymerase
• These s factors change the host polymerase
specificity of promoter recognition from
early to middle to late
– The host s factor is specific for the phage
early genes
– Phage gp28 protein switches the specificity to
the middle genes
– Phage gp33 and gp34 proteins switch to late
specificity
Sporulation芽胞產生
•During infection, phage SPO1
changes specificity of host RNA
polymerase
•Same type of mechanism
applies to changes in gene
expression during sporulation
–Bacteria can exist indefinitely in
vegetative state營養期 if
nutrients are available
–Under starvation conditions, B.
subtilis forms endospores,
though dormant bodies
Vegetative cell
營養細胞
Endospore 內孢子
Sporulating cell 孢子細胞
Sporulation Switching
•During sporulation, a whole new set of genes is
turned on, and vegetative genes are turned off
•Switch occurs largely at the level of transcription
•Several new s-factors displace the vegetative sfactor from the polymerase core
•Each s-factor has its own preferred promoter
sequence
Vegetative cells: sA
Appears first, activates transcription of the
recognize
promoterss-factors
like E. coli
other
sporulation-specific
-10 box (TATAAT) and -35 box (TTGACA)
Sporulating cells: sF, sE, sH, sC, sK
different promoter sequence
Vegetative promoter
sporulation promoter
Genes With Multiple Promoters
• Some genes must be expressed during two or
more phase of sporulation
• Multiple promoters recognized by the different
s-factor
Sporulating cells: sF,sE,sH, sC, sK
Vegetative cells: sA
recognize promoters like E. coli
P1: recognized by sB
P2: recognized by sE
Overlapping promoters in B. subtilis spo VG gene.
Other s-Switches
Bacterial Heat Shock
• The heat shock response 熱休克反應 is a defense
by cells to minimize damage
• Molecular chaperones分子伴護蛋白are proteins:
– Bind proteins partially unfolded by heating
• Help these proteins refold properly
– Proteases
• Degrade proteins that can not be refold
• Genes encoding proteins that help cells survive
heat shock are called heat shock genes
Other s-Switches
•Heat shock response is governed by an
alternative s-factor, s32 or sH (H: heat shock)
–Directs RNA polymerase to the heat shock gene
promoters
–Accumulation of sH with high temperature is due
to:
• Stabilization of sH
• Enhanced translation of the mRNA encoding sH
•Responses to low nitrogen and starvation stress
also depend on genes recognized by other sfactors (sN, sS )
8.2 The RNA Polymerase
Encoded in Phage T7
• Phage like T7, T3, and 11 have small genomes and
many fewer genes
• These phage have 3 phases of transcription:
classes I, II, and III
• Of 5 class I genes, gene 1 is necessary for class II and
class III gene expression
– If gene 1 is mutated, only class 1 genes are
transcribed
– Gene 1 codes for a phage-specific RNA
polymerase of just one polypeptide
Temporal Control
of Transcription
•Host polymerase
transcribes the class I
genes
•Gene 1 RNA
5
polymerase transcribes
only T7 class II and III
genes, not class I genes
Host RNA polymerase
genes, gene1: RNA polymerase
8.3 Infection of E. coli by Phage 
• Virulent phage活性噬菌體 (致病性) replicate and
kill their host by lysing 溶解 or breaking it
open, such as T7, SPO1
• Temperate phage溫和性噬菌體, such as ,
infect cells but don’t necessarily kill 侵入細菌
後不立刻導致增殖、溶菌，可與菌體共生的噬菌體
– The temperate phage have 2 paths of
reproduction
• Lytic mode 溶解性: infection progresses as in
a virulent phage 感染進程似致病性噬菌體
• Lysogenic mode 潛溶性: phage DNA is
integrated into the host genome 噬菌體DNA會
嵌插入寄主的基因體內
Lytic versus lysogenic infection by phage 
Lytic phase
Lysogenic phase
Lytic versus lysogenic infection by phage 
Lytic phase
•噬菌體DNA進入寄主細胞(細菌)
•以此DNA當作模板,並利用寄主的
RNA polymerase 進行轉錄
•噬菌體的 mRNAs 轉譯成噬菌體
的蛋白質
•噬菌體 DNA 經複製後, 藉由噬菌
體蛋白質進行組合,將寄主溶解
是釋出噬菌體
Lytic versus lysogenic infection by phage 
Lysogenic phase
• 有一 27-kD 噬菌體蛋白 (稱之
為 repressor, CI)可與噬菌體
的兩個opertor結合
• CI 除了本身基因外,會使所有
的基因不再轉錄
• Lysogen 潛溶性是指細菌包含
具有鑲嵌噬菌體DNA的基因體
 稱之為prophage
•Lysogenic phase lytic
phase
prophage
Lytic Reproduction of Phage 
• 噬菌體的溶解期 (Lytic reproduction cycle)
依其轉錄的時序分為三個時期:
– Immediate early 早初期
– Delayed early 晚初期
– Late 晚期
• 各時期基因依序排列在噬菌體的DNA
Genetic Map of Phage 
Linear form 直線型
依其功能分組
circle form
圓形
•在噬菌體內DNA以直線形
存在
•當噬菌體感染細菌時,噬
菌體DNA會成圓形
– 直線型兩端具cos端,可互
相黏合
Cohesive end
12bp
Antitermination 抗終止
• Antitermination 抗終止作用屬一種轉錄的轉換機
制 (transcriptional switch)
• 有些基因的產物當作抗終止因子(antiterminator)
– 使得RNA polymerase 忽略早初期基因
(intermmediate early genes)的終止訊息而繼續轉錄
晚初期基因
– 故使同一個啟動子可以同時用於轉錄早初期及晚初
期基因
– 另一抗終止因子則因忽略終止訊息而促使晚期基因
的表現
Antitermination and Transcription抗終止及轉錄
•Immediate early genes 早初期基因
–cro codes for a repressor 本身產物是一抑制子
• 此蛋白質的合成主要是抑制  抑制性基因 (repressor gene, cI) 的轉
錄而生成抑制性蛋白
–N gene coding for N, an antiterminator 為抗終止因子
• 使得RNA polymerase 忽略早初期基因的終止訊息而繼續轉錄晚初
期基因
•無論immediate early 及 delayed early的轉錄均利用相同的啟
動子 (PR and PL)
由寄主的全酶 (holoenzyme)
進行早初期基因的轉錄
Antitermination and
Transcription
•Delayed early genes 晚初期基因
– 利用相同的啟動子 (PR and PL)
Delayed early genes
– Gene O 及 P 與 phage DNA 複製有關
(lytic growth)
– Q gene (antiterminator為抗終止因子) 使
得繼續轉錄晚期基因
•Late genes 晚期基因
– 利用晚期啟動子late
promoter, PR’,
– 製造有關於噬菌體的頭及尾
的蛋白質,最終將細菌溶解
No new s-factor or RNA
polymerase involved 沒有
新的 s-factor及RNApolymerase參與轉錄
Late genes
N Antitermination
Function
• N gene 附近包含:
–向左的啟動子 (Left promoter, PL)
–操作子 (Operator, OL )
–轉錄終止子 (Transcription terminator)
(不具 N 終止termination)
• When N is present當N存在時:
–N 蛋白會與mRNA上的 N utilization
site (nut site) 結合
–同時還會與 polymerase 及寄主蛋白
質作用
–這樣的結構使得Polymerase 忽略正常
的終止子,而繼續轉錄晚初期基因
Proteins Involved in NDirected Antitermination
在早初期之抗終止作用需寄主五個蛋白質的幫忙
– NusA 及ribosomal S10 會與RNA polymerase 結合
– N 及 NusB 會與nut site 的 box B 及box A 作用
– N 及 NusB 將轉錄的基因與NusA 及 S10 及
polymerase 結合
•NusA 本身可促進轉錄的終止
Proteins Complexes Involved in
N-Directed Antitermination
Slow down hairpin formation
Host Proteins:
 anti-termination
NusA
NusB
NusG
S10
Antitermination and Q
• 在晚期基因的抗終止作用則須 Q
• Q protein會與Q-binding region of the qut site (Q
utilization site) 結合,使得RNA polymerase 停留在
late promoter (PR’) 下游
• 當 Q 與 polymerase結合時會改變酵素的構造進而
忽略終止子而持續轉錄晚期基因
Establishing Lysogeny 建立潛溶期
由兩個啟動子控制 cI gene:
PRM: promoter for repressor maintenance (維持)
PRE: promoter for repressor establishment (建立)
•No consensus sequence with -10 and -35 box
• cII gene 產物幫助RNA polymerase 與獨特的啟動子結合
(包括PRE and PI (I: int))
•造成兩種影響:
• 產生Antisense cro gene – 會與sense cro gene結合
干擾cro gene的轉錄
促進形成潛溶期
• cI gene –產生抑制子repressor
Establishing Lysogeny
• 潛溶期的建立乃因:
– 產生抑制性蛋白 (repressor)與早期操作子結合
– 避免早期的 RNA合成
• 此時後期基因的表現則用於
– 鑲嵌入寄主的基因體內
– 產生 cII 及 cIII 產物使得產生更多的 cI gene的產物亦即製造更
多的  repressor
• CII protein 會促使RNA polymerase 與PRE 結合以轉錄 cI gene, 產
生更多抑制性蛋白
• 建立前溶期的最主要啟動子是 PRE
Autoregulation of the cI
Gene During Lysogeny
• 當  repressor出現時,會以 dimer 的型式與 
operators, OR and OL 結合:
– Repressor 關掉早期的轉錄作用
• 破壞 lytic cycle
• 關閉 cro gene的表現
– product Cro acts to counter repressor activity
– 藉由活化 activating PRM來刺激本身蛋白質的
形成
cro : gene
Cro: protein
Repressor Protein
Repressor protein
– A dimer of 2 identical subunits
– Each subunit has 2 domains with distinct roles
• Amino-terminal is the DNA-binding end of
molecule
• Carboxyl-terminal is site of repressorrepressor interaction that makes dimerization
and cooperative binding possible
Maintaining Lysogeny
Self-activation
• Repressor block cII and cIII
transcription (PRE)
• Block cro (PR)
• Repressor binds most
tightly to OR1
• Binding of repressor to OR1
and OR2 is cooperative
operator
Model of Involvement
of OL in Repression of
PR and PRM
Repression of PR
Repression of PRM
當抑制子太多時,會與OR3結
合, 而抑制PRM
Involvement of OL in Repression
• Repressor binds to OR1 and OR2 cooperatively, but
leaves OR3
• RNA polymerase to PRM which overlaps OR3 in such
a way it contacts repressor bound to OR2
• Protein-protein interaction is required for promoter
to work efficiently
• High levels of repressor can repress transcription
from PRM
– Process may involve interaction of repressor dimers
bound to OR1, OR2, and OR3
– Repressor dimers bound to OL1, OL2, and OL3 via
DNA looping
Activation Via Sigma
• Promoters subject to
polymerase-repressor
activation have weak 35 boxes
• These boxes are poorly
recognized by s
• Activator site overlaps 35 box, places activator
in position to interact
with region 4
Determining the Fate of a 
Infection
• Balance between lysis or lysogeny is delicate
• Place phage particles on bacterial lawn
– If lytic infection occurs
• Progeny spread and infect other cells
• Circular hole seen in lawn is called plaque
– Infection 100% lytic gives clear plaque
– Plaques of  are usually turbid meaning live
lysogen is present
• Some infected cells suffer the lytic cycle, others are
lysogenized
Battle Between
cI and cro
•The cI gene codes for
repressor, blocks OR1, OR2,
OL1, and OL2 so turning off
early transcription
leads to lysogeny
•The cro gene codes for Cro
that blocks OR3 and OL3,
turning off transcription
 leads to lytic infection
•Gene product in high
concentration first
determines cell fate
Lysogen
Induction
•When lysogen suffers DNA
damage, SOS response is
induced
•Initial event is seen in a
coprotease activity in RecA
protein
•Repressors are caused to
cut in half, removing them
from  operators
•Lytic cycle is induced
•Progeny phage can escape
potentially lethal damage
occurring in host