Chapter 6 The mechanism of transcription in prokaryotes
Chapter 6 The mechanism of transcription in prokaryotes
DNA aooogo Replication 叶 DNA duplicates NNV八X000a0 N八V八aa八NN DNA Transcription RNA synthesis RNA MRNA nucleus cytoplasm nuclear envelope T Translation Protein synthesis Protein Protein The Central Dogma of Molecular Biology
Content Transcription:Initiation Elongation carried out by RNA Polymerase Termination binding Promoter
Transcription: Initiation Elongation Termination binding carried out by RNA Polymerase Promoter Content
6.1 RNA polymerase structure 2Xa subunit (40kD) Core enzyme 】 B subunit RNA polymerase (150kD) (Holoenzyme) B'subunit subunit (160kD) Figure 6.1 (70kD) Holoenzyme Core The subunit content of an RNA polymerase holoenzyme(全酶)is β，β，o,02 The RNA polymerase without o-subunit is called the core polymerase(核心酶) 2 3
6.1 RNA polymerase structure RNA polymerase (Holoenzyme) Core enzyme 2Xa subunit b subunit b’subunit s subunit (150kD) (160kD) (40kD) (70kD) The subunit content of an RNA polymerase holoenzyme(全酶) is β',β,σ,α2 The RNA polymerase without σ-subunit is called the core polymerase（核心酶）
Transcription activity of core and holoenzyme Figure T6.1 Table 6.1 Ability of Core and Holoenzyme to Transcribe DNAs Relative Transcription Activity DNA Core Holoenzyme T4(native,intact) 0.5 33.0 Calf thymus(native,nicked) 14.2 32.8 o-subunit was very important to the enzyme's activity. what is the o-subunit's function?
Transcription activity of core and holoenzyme σ-subunit was very important to the enzyme's activity. what is the σ-subunit’s function?
Sigma(o)as a Specificity Factor Immediate early (0-2min) Delayed early (2-10min) 立即早期 延迟早期 3 Late(10-25min)） Holoenzyme 晚期 Immediate early (0-2min) Core enzyme o factor confers specificity to the polymerase. o directs polymerase to initiate at specific sites,called promoters. How can we confirm the o-subunit's function by experiment?
5’ 3’ 3’ 5’ Immediate early (0-2min) 立即早期 Delayed early (2-10min) 延迟早期 Late (10-25min) 晚期 Immediate early (0-2min) 5’ 3’ 3’ 5’ Holoenzyme Core enzyme 5’ 3’ 3’ 5’ s factor confers specificity to the polymerase. s directs polymerase to initiate at specific sites, called promoters. How can we confirm the σ-subunit’s function by experiment? Sigma (σ) as a Specificity Factor
Copyright:The McGraw-Hill Companies,Inc.Permission required for reproduction or display. X Y Denature DNA Transcription of region X with labeled NTPs (大NTP) immobilized Transcript base-pairs with Template on a support (a) Hybridization with no competitor bHybridization with competitor Unlabeled ribonuclease(RNase) competitor X RNAS Measure labeled hybrids Measure labeled hybrids saturation level Y RNA competitor Label Label n hybrid hybrid saturating X RNA competitor Amount of labeled amount of Amount of unlabeled RNA added labeled RNA cmpetitor RNA added Figure6.2 Hybridization-.competition(竞争杂交)
Figure 6.2 Hybridization-competition（竞争杂交） Transcript base-pairs with Template immobilized on a support ribonuclease (RNase) saturation level saturating amount of labeled RNA
Copyright The McGraw-Hill Companies,Inc.Permission required for reproduction or display. 100 The transcripts made by the core (a)Core polymerase can be competed to 75 immediate early about the same extent by immediate early,delayed early, 50 and late T4 RNAs.The core delayed early 25 enzyme has no specificity △△ late △ 100 (b)Holoenzyme 75 The transcripts made by the holoenzyme can be competed to a great extent by T4 immediate early RNA. 50 The holoenzyme is 25 highly specific for the immediate 250 500 7501000 early genes Competitor RNA added (ug) Figure 6.3 The o factor confers specificity for the T4 immediate early genes
Figure 6.3 The σ factor confers specificity for the T4 immediate early genes. The holoenzyme is highly specific for the immediate early genes The transcripts made by the core polymerase can be competed to about the same extent by immediate early, delayed early, and late T4 RNAs.The core enzyme has no specificity immediate early delayed early late The transcripts made by the holoenzyme can be competed to a great extent by T4 immediate early RNA
SUMMARY The key player in the transcription process is RNA polymerase.The E.coli enzyme is composed of a core,which contains the basic transcription machinery,and a o-factor,which directs the core to transcribe specific genes
SUMMARY The key player in the transcription process is RNA polymerase. The E.coli enzyme is composed of a core, which contains the basic transcription machinery, and a σ–factor, which directs the core to transcribe specific genes
Figure T6.1 Table 6.1 Ability of Core and Holoenzyme to Transcribe DNAs Relative Transcription Activity DNA Core Holoenzyme T4(native,intact) 0.5 33.0 Calf thymus(native,nicked) ★14.2 32.8 why was core RNA polymerase still capable of transcribing nicked DNA,but not intact DNA? Nicks and gaps in DNA provide ideal initiation sites for RNA polymerase (nonspecific when o was present,the holoenzyme could recognize the authentic RNA polymerase binding sites and begin transcription. Promoter
why was core RNA polymerase still capable of transcribing nicked DNA, but not intact DNA? Nicks and gaps in DNA provide ideal initiation sites for RNA polymerase (nonspecific ) when σ was present, the holoenzyme could recognize the authentic RNA polymerase binding sites and begin transcription. Promoter