Molecular Biology Part I: Chemistry and Genetics Part II: Maintenance of the Genome Part III: Expression of the Genome Part IV: Regulation Part V: Methods
Part IV Regulation Ch 16: Regulation in prokaryotes Ch 17: Regulation in eukaryotes Ch 18: Regulation during development and in diseases (brief introduction) Ch 19: Comparative genomics and evolution of animal diversity(Not covered in the lecture)
Expression of many genes in cells are regulated Housekeeping genes: expressed constitutively, essential for basic processes involving in cell replication and growth. Inducible genes: expressed only when they are activated by inducers or cellular factors.
Chapter 16 Regulation principles and How genes are regulated in bacteria Chapter 17 Basic mechanism of gene expression in eukaryotes Chapter 18 The mechanism of RNAi and the role of miRNA in development and cancergenesis
Surfing the contents of Part IV --The heart of the frontier biological disciplines
Some of the peoples who significantly contribute to the knowledge of gene regulation
Chapter 16 Gene Regulation in Prokaryotes • Molecular Biology Course
TOPIC 1Principles of Transcriptional Regulation [watch the animation] TOPIC 2 Regulation of Transcription Initiation: Examples from Bacteria (Lac operon, alternative s factors, NtrC,MerR, Gal rep, araBAD operon) TOPIC 3 Examples of Gene Regulation after Transcription Initiation (Trp operon) TOPIC 4 The Case of Phage λ: Layers of Regulation
CHAPTER 16 Gene Regulation in Prokaryotes Topic 1: Principles of Transcription Regulation
Principles of Transcription Regulation 1. Gene Expression is Controlled by Regulatory Proteins (调控蛋白) Gene expression is very often controlled by Extracellular Signals,which are communicated to genes by regulatory proteins: • Positive regulators or activators INCREASE the transcription • Negative regulators or repressors DECREASE or ELIMINATE the transcription
Principles of Transcription Regulation 2. Gene expression is controlled at different stages(基因表达可以发生在不同时期) • The bulk of gene regulation takes place at the initiation of transcription. • Some involve transcriptional elongation/termination, RNA processing, and translation of the mRNA into protein.
Fig 12-3-initiation Promoter Binding (closed complex) Promoter “melting” (open complex) Promoter escape/Initial transcription
Fig 12-3-Elongation and termination Elongation Termination
Principles of Transcription Regulation 3. Targeting promoter binding: many promoters are regulated by activators(激活蛋白)that help RNAP bind DNA (recruitment) and by repressors(阻遏蛋白)that block the binding. RNAP binds many promoters weakly (?), activators that contain two binding sites to bind a DNA sequence and RNAP simultaneously can enhance the RNAP affinity with the promoters, and thus increases gene transcription.This is called recruitment regulation (招募调控). On the contrary, Repressors can bind to the operator inside of the promoter region, which prevents RNAP binding and the transcription of the target gene.
Fig 16-1 a. Absence of Regulatory Proteins: basal level expression b. Repressor binding to the operator represses expression c. Activator binding activates expression
Principles of Transcription Regulation 4 Targeting transition to the open complex: Allostery regulation (异构调控)after the RNA Polymerase Binding In some cases, RNAP binds the promoters efficiently, but no spontaneous isomerization occurs to lead to the open complex, resulting in no or low transcription. Some activators can bind to the closed complex, inducing conformational change in either RNAP or DNA promoter, which converts the closed complex to open complex and thus promotes the transcription.
Allostery regulation Fig 16-2 Allostery is not only a mechanism of gene activation , it is also often the way that regulators are controlled by their specific signals.
Principles of Transcription Regulation 5 Targeting promoter escape by some repressors Repressors can work in ways: blocking the promoter binding. blocking the transition to the open complex. blocking promoter escape
Some promoters are inefficient at more than one step and can be activated by more than one mechanism. Activation mechanisms include recruitment(招募)and allostery (异构).
6. Cooperative binding (recruitment) and allostery have many roles in gene regulation Principles of Transcription Regulation For example: group of regulators often bind DNA cooperatively (activators and/or repressors interact with each other and with the DNA, helping each other to bind near a gene they regulated) : produce sensitive switches to rapidly turn on a gene expression, integrate signals (some genes are activated when multiple signals are present).
Principles of Transcription Regulation 7.Action at a Distance and DNA Looping. The regulator proteins can function even binding at a DNA site far away from the promoter region, through protein-protein interaction and DNA looping. Fig 16-3
Fig 16-4 DNA-binding protein can facilitate interaction between DNA-binding proteins at a distance Fig 16-4
CHAPTER 16 Gene Regulation in Prokaryotes Topic 2: Regulation of Transcription Initiation : Examples from Bacteria
Operon:a unit of prokarytoic gene expression and regulation which typically includes: 1.Structural genesfor enzymes in a specific biosynthetic pathway whose expression is coordinately controlled. 2.Control elements, such as operator sequence. 3.Regulator gene(s)whose products recognize the control elements. Sometimes are encoded by the gene under the control of a different promoter
Control element Structural genes
Regulation of Transcription Initiation in Bacteria First example: Lac operon The lactose Operon (乳糖操纵子)
1. Lactose operon containsa regulatory gene and 3 structural genes, and 2 control elements. Fig 16-5 The enzymes encoded by lacZ, lacY, lacA are required for the use of lactose as a carbon source. These genes are only transcribed at a high level when lactose is available as the sole carbon source. The LAC operon
codes for β-galactosidase (半乳糖苷酶) for lactose hydrolysis lacZ encodes a cell membrane protein called lactose permease (半乳糖苷渗透酶) to transport Lactose across the cell wall lacY encodes a thiogalactoside transacetylase (硫代半乳糖苷转乙酰酶)to get rid of the toxic thiogalacosides lacA The LAC operon
The lacZ, lacY, lacA genes are transcribed into a single lacZYA mRNA (polycistronic mRNA) under the control of a signal promoter Plac. LacZYA transcription unit contains anoperator site Olac position between bases -5 and +21 at the 3’-end of Plac Binds with the lac repressor The LAC operon
2. An activator and a repressor together control the Lac operon expression The activator:CAP (Catabolite Activator Protein,代谢产物激活蛋白) or CRP (cAMP Receptor Protein,cAMP受体蛋白); responses to the glucose level. The repressor:lac repressor that is encoded by LacIgene; responses to the lactose. Sugar switch-off mechanism The LAC operon
The LAC operon Fig 16-6
The LAC operon 3. Lac repressor bound to the operator prevents RNAP from binding to the promoter The site bound by lac repressor is called the lac operator (Olac), and the Olacoverlaps promoter (Plac). Therefore repressor bound to the operator physically prevents RNA polymerase from binding to the promoter. The LAC operon
The LAC operon Fig 16-8
The LAC operon 4. CAP activates the Lac transcription through recruitment of RNAP to the weak Plac CAP has two binding sites, one interacts with the CAP site on the DNA near promoter, and one interacts with RNAP. This cooperative binding ensures that RNAP effectively binds to Plac and initiates transcription of LacZYA. The LAC operon
CAPsite has the similar structure as the operator, which is 60 bp upstream of the start site of transcription. • CAP also interacts with the RNAP and recruit it to the promoter. Fig 16-9 a CTD: C-terminal domain of the a subunit of RNAP
The LAC operon CAP binds as a dimer a CTD Fig 16-10. CAP has separate activating and DNA-binding surface
5. CAP and Lac repressor bind DNA using a common structural motif: helix-turn-helix motif Fig 16-11 One is the recognition helix that can fits into the major groove of the DNA. The LAC operon
DNA binding by a helix-turn-helix motif Fig 16-12 Hydrogen Bonds between l repressor and the major groove of the operator.
Lac operon contains three operators: the primary operator and two other operators located 400 bp downstream and 90 bp upstream. Lacrepressor binds as a tetramer (四聚体), with each operator is contacted by a repressor dimer (二聚体). respectively. Fig 16-13
6 The activity of Lac repressor and CAP are controlled allosterically by their signals. Allolactose: turn of Lac repressor cAMP: turn on CAP Lactose is converted to allolactose by b-galactosidase, therefore lactose can indirectly turn off the repressor. Glucose lowers the cellular cAMP level, therefore, glucose indirectly turn off CAP. The LAC operon
Absence of lactose z y a i p o Active Very low level of lac mRNA Response to lactose Lack of inducer: the lac repressor block all but a very low level of trans-cription of lacZYA . When Lactose is present, the low basal level of permease allows its uptake, and b-galactosidase catalyzes the conversion of some lactose to allolactose. Allolactoseacts as an inducer, binding to the lac repressor and inactivate it. Presence of lactose z y a i p o Inactive Permease Transacetylase b-Galactosidase
7: Combinatorial Control (组合调控): CAP controls other genes as well • A regulator (CAP) works together with different repressor at different genes, this is an example of Combinatorial Control. • In fact, CAP acts at more than 100 genes in E.coli, working with an array of partners.
Regulation of Transcription Initiation in Bacteria Second example: Alternative s factor Alternative s factor (可变s因子)direct RNA polymerase to alternative site of promoters
factor subunit bound to RNA polymerase for transcription initiation (Ch 12)
Different factors binding to the same RNAP, conferring each of them a new promoter specificity. • 70factors is most common one in E. coli under the normal growth condition
Many bacteria produce alternative sets of σfactors to meet the regulation requirements of transcription under normal and extreme growth condition. Bacteriophage has its own σfactors E. coli : Heat shock 32 Bacteriophage σfactors Sporulation in Bacillus subtilis
Heat shock (热休克) • Around 17 proteins are specifically expressed in E. coli when the temperature is increased above 37ºC. • These proteins are expressed through transcription by RNA polymerase using an alternative factor 32 coded by rhoH gene. 32has its own specific promoter consensus sequences. Alternative s factors