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  switches The RNA polymerase Encoded in phage T7 Infection of E.coli by phage –Lytic Reproduction of phage –Establishing Lysogeny

轉換 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  is the key factor in determining specificity of T4 DNA transcription To shift the transcription process  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 2 , ,  ’, ,  (43kDa),  (prevent binding to nonpromoter region)

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 Temporal Control of Transcription gp28: new  factor Phage  factor Host core polymerase gp33, gp34: another  factor Phage gp33, gp34 Host core polymerase Material: Phage SPO1-infected B. subtilis change specificity Transcribe phage gene From early to middle phage transcription  : 43kDa

phage- encoded  factors host core RNA polymeraseThis switching is directed by a set of phage- encoded  factors that associate with the host core RNA polymerase These  factors change the host polymerase specificity of promoter recognition from early to middle to late –The host  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 Transcription Switching

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 Sporulating cell 孢子細胞 Endospore 內孢子

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  -factors displace the vegetative  - factor from the polymerase core Each  -factor has its own preferred promoter sequence Sporulation Switching Vegetative cells:  A recognize promoters like E. coli -10 box (TATAAT) and -35 box (TTGACA) Sporulating cells:  F,  E,  H,  C,  K different promoter sequence Vegetative promoter sporulation promoter Appears first, activates transcription of the other sporulation-specific  -factors

Genes With Multiple Promoters Genes With Multiple Promoters Sporulating cells:  F  E  H  C  K Vegetative cells:  A recognize promoters like E. coli Overlapping promoters in B. subtilis spo VG gene. P1: recognized by  B P2: recognized by  E Some genes must be expressed during two or more phase of sporulation Multiple promoters recognized by the different  -factor

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  -Switches

Heat shock response is governed by an alternative  -factor,  32 or  H (H: heat shock) –Directs RNA polymerase to the heat shock gene promoters –Accumulation of  H with high temperature is due to: Stabilization of  H Enhanced translation of the mRNA encoding  H Responses to low nitrogen and starvation stress also depend on genes recognized by other  - factors (  N,  S ) Other  -Switches

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 phage-specific RNA polymerase –Gene 1 codes for a phage-specific RNA polymerase of just one polypeptide

Temporal Control of Transcription Host RNA polymerase 5 genes, gene1: RNA polymerase Host polymerase transcribes the class I genes Gene 1 RNA polymerase transcribes only T7 class II and III genes, not class I genes

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 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 Linear form 直線型 circle form 圓形 依其功能分組

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 抗終止

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 (P R and P L ) 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 (P R and P L ) –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, P R’, – 製造有關於噬菌體的頭及尾 的蛋白質, 最終將細菌溶解 Delayed early genes Late genes No new  -factor or RNA polymerase involved 沒有 新的  -factor 及 RNApolymerase 參與轉錄

N Antitermination Function N gene 附近包含 : – 向左的啟動子 (Left promoter, P L ) – 操作子 (Operator, O L ) – 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

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 Involved in N- Directed Antitermination

Proteins Complexes Involved in N-Directed Antitermination Host Proteins: NusA NusB NusG S10 Slow down hairpin formation  anti-termination

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 (P R ’ ) 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 Antitermination and Q

Establishing Lysogeny 建立潛溶期 由兩個啟動子控制 cI gene: P RM : promoter for repressor maintenance ( 維持 ) P RE : 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 P RE and P I (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 P RE 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 P RE

當 repressor 出現時, 會以 dimer 的型式與 operators, O R and O L 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 P RM 來刺激本身蛋白質的 形成 Autoregulation of the cI Gene During Lysogeny cro : gene Cro: 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 repressor- repressor interaction that makes dimerization and cooperative binding possible Repressor Protein

Maintaining Lysogeny Self-activation Repressor binds most tightly to O R1 Binding of repressor to O R1 and O R2 is cooperative operator Repressor block cII and cIII transcription (P RE ) Block cro (P R )

Model of Involvement of O L in Repression of P R and P RM Repression of P R Repression of P RM 當抑制子太多時, 會與 O R 3 結 合, 而抑制 P RM

Involvement of O L in Repression Repressor binds to O R 1 and O R 2 cooperatively, but leaves O R 3 RNA polymerase to P RM which overlaps O R 3 in such a way it contacts repressor bound to O R 2 Protein-protein interaction is required for promoter to work efficiently High levels of repressor can repress transcription from P RM –Process may involve interaction of repressor dimers bound to O R 1, O R 2, and O R 3 –Repressor dimers bound to O L 1, O L 2, and O L 3 via DNA looping

Activation Via Sigma Promoters subject to polymerase-repressor activation have weak - 35 boxes These boxes are poorly recognized by  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 O R 1, O R 2, O L 1, and O L 2 so turning off early transcription  leads to lysogeny The cro gene codes for Cro that blocks O R 3 and O L 3, 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