2. Section L: Regulation of transcrip. in Prok.Yang Xu, College of Life Sciences Section L Regulation of Transcription in Prokaryotes L1 The lac operon L2 The trp operon L3 Transcriptional regulation by alternative  factor

3. Section L: Regulation of transcrip. in Prok.Yang Xu, College of Life Sciences L1 The lac operon The operon The lactose operon The lac repressor Induction cAMP receptor protein

4. Francois Jacob (44y) Jacques Monod (55y) (French) Lac. Operon Theory Nobelists in Biology in 1965 Concept of mRNA Francois Jacob Jacques Monod

5. Section L: Regulation of transcrip. in Prok.Yang Xu, College of Life Sciences The operon Definition: The operon is a unit of gene expression and regulation which typically includes: Regulator genes: whose products recognize the control-elements, for example a repressor which binds to and regulates an operator sequence. Operator sequence: Control elements such as an operator sequence, which is a DNA sequence that regulates transcription of the structural genes. Structural genes: The structural genes for encoding proteins. Structural genes Regulator genesOperator sequence P lacI lacIP lac O lac lacYlacAlacZ regulationinformation

6. Section L: Regulation of transcrip. in Prok.Yang Xu, College of Life Sciences The lactose operon Structural genes: The lactose operon consists of 3 structural genes: lacZ, lacY, lacA. Polycistronic mRNA: The three structural genes are encoded in a single transcription unit lacZYA, which has a single promoter P lac, and transcribes a single polycistronic mRNA, but more proteins expressed.

7. Section L: Regulation of transcrip. in Prok.Yang Xu, College of Life Sciences The lac repressor Definition:  The lac I gene encodes the repressor,  which is active as a tetramer of identical subunits.  It has a very strong affinity for the lac operator-binding site, O lac, and  also has a generally high affinity for DNA. Structure:  The lac operator-binding site consists of 28 bp  which is palindromic.  This inverted symmetry of the operator matches the inherent symmetry of the lac repressor. DNADNA

8. Section L: Regulation of transcrip. in Prok.Yang Xu, College of Life Sciences The lac repressor Binding:  In the absence of lactose, the repressor occupies the operator-binding site.  The lac repressor increases the binding of the RNA polymerase to the lac promoter by two order of magnitude.  This means that when lac repressor is bound to the O lac, the RNA plo is also likely to be bound to adjacent P lac promoter sequence without move.

9. Section L: Regulation of transcrip. in Prok.Yang Xu, College of Life Sciences P lacI lacIP lac O lac lacYlacAlacZ Induction process RNAp Low Lac. RNAp mRNA 4. Transcription: 5. Re-Inhibition: 1. No lactose: In the absence of lactose, the lac repressor blocks all but a very low level of transcription of lacZYA. 2. Uptake: When lactose is added to cell, the low level of permease allows its uptake, and  galactosidases catalyzes some lactose to allo-lactose. 3. Induction: Allo-lactose acts as an inducer and binds to the lac represser. This causes a change in the conformation of the repressor tetramer, reducing its affinity for lac operator. RNAp

11. Section L: Regulation of transcrip. in Prok.Yang Xu, College of Life Sciences cAMP receptor protein-I Function: The P lac promoter is not a strong promoter. P lac and related promoters do not have strong -35 sequences and some even have weak -10 consensus sequences. For high level transcription, they require the activity of a specific protein called cAMP receptor protein (CRP). CRP exists as a dimer which cannot bind to DNA on its own, nor regulate transcription, but which can form CRP-cAMP

12. CRP-cAMP ( cAMP receptor protein ) binding site ● CRP-cAMP binds site I ● site I : IR is CRP-cAMP strong binding site （ -70 ~ -50 ） ● site II: is CRP-cAMP weak bingding site （ -50 ~ -40 ） cooperative effect Promote site II binding IR -70 site I -40 site II IR -50

13. SiteI siteII GC Island -35 -10 ● CRP-cAMP + site II promotion RNApol. into -35 sequence into -10 sequence starting transcription RNApol

14. Section L: Regulation of transcrip. in Prok.Yang Xu, College of Life Sciences cAMP receptor protein-II Normal Condition: The glucose is present in E. coli. 1. Inactivated operon: The E. coli does not require alternative carbon sources such as lactose. Therefore lactose operon is normally inactivated. 2. Inactivated CRP: The glucose can reduce the level of cAMP, therefor the cAMP is not enough for binding CRP. Special Condition: When glucose is absent in E. coli culture. 1. Activated CRP: The levels of cAMP increased, and CRP binds to cAMP to form the CRP-cAMP complex which is the activated CRP. 2. Activated operon: The CRP- cAMP complex binds to the P lac just upstream from the site for RNA pol. CRP binding induces a 90º bend in DNA, and to enhance RNA pol binding to the promoter, enhancing transcription by 50-fold.

15. Section L: Regulation of transcrip. in Prok.Yang Xu, College of Life Sciences L2 The trp operon The tryptophan operon The try repressor The attenuator Leader RNA structure Attenuation Importance of attenuation trp

16. Section L: Regulation of transcrip. in Prok.Yang Xu, College of Life Sciences The tryptophan operon Structural genes: The trp operon encodes five structural genes whose activity is required for tryptophan synthesis. Transcript: The operon encodes a single transcription unit which produces a 7 kb transcript which is synthesized downstream from the trp promoter P try and trp operator sites O trp. Expression: Like many of the operons involved in amino acid biosynthesis, the trp operon has evolved systems for co- ordinated expression of these genes when tryptophan is in short supply in the cell. Regulation speed: As with the lac operon, the RNA product of this transcription unit is very unstable, enabling bacteria to respond rapidly to changing needs for tryptophan.

17. trpR P O E D C B A tryptophan Mechanism Operator operon off operon on repressor + trp active repressor Repressor (inactive ) can not bind on the O site (trp absent)

18. Section L: Regulation of transcrip. in Prok.Yang Xu, College of Life Sciences The trp repressor Trp repressor: A gene product of the trpR operon. It is a dimer of two subunits. Operator structure: P trp is between -21 and +3. The core binding site is a palindrome of 18bp. Mechanism:  The trp repressor can only bind to the operator when it is complexed with tryptophan.  The repressor dimer has a structure with a central core and two DNA-reading heads.  When tryptophan is bound to the repressor the reading heads are the correct distance apart, and the side chains in the correct conformation, to interact with major grooves of the DNA at P trp. Tryptophan: is the end-product of the enzymes encoded by the trp operon, it acts as a co- repressor and inhibits its own synthesis by end- product inhibition. The repressor reduces transcription initiation by around 70-fold. trp

19. Section L: Regulation of transcrip. in Prok.Yang Xu, College of Life Sciences

20. Section L: Regulation of transcrip. in Prok.Yang Xu, College of Life Sciences The attenuator Background: At first, it was thought the repressor was responsible for all of the transcriptional regulation of the trp operon. And then, it was observed that the deletion of a sequence between the operator and the trpE resulted in an increase in both the basal and the activated (de-repressed) levels of transcription. Attenuator: This site is termed the attenuator and it lies towards the end of the transcribed leader sequence of 162 nt that precedes the trpE initiator codon. Structure: The attenuator is a  -independent terminator site which has a short GC-rich palindrome followed by eight successive U residues in RNA sequence. Function: If this sequence is able to form a 3 － 4 hairpin structure in the RNA transcript, then it acts as a highly efficient transcription terminator and only a 140 nt transcript is synthesized.

21. Section L: Regulation of transcrip. in Prok.Yang Xu, College of Life Sciences S3S4 A A U U G C A C G G C C G G C A U ……NNNN UUUUUUU-OH Rho-independent T.

22. Section L: Regulation of transcrip. in Prok.Yang Xu, College of Life Sciences

23. Section L: Regulation of transcrip. in Prok.Yang Xu, College of Life Sciences Attenuation Reason of attenuation: Attenuation effect depends on the fact that transcription and translation are tightly coupled in E. coli; translation can occur as an mRNA is being transcribed. Sequence 1:  The 3’OH-end of the trp leader peptide coding sequence overlaps complementary sequence 1;  the two trp codons are within sequence 1. Stop codon: The stop codon in the leader sequence is between sequence 1 and sequence 2. 3:4 hairpin: It is a conditional terminator (attenuator). When the tryptophan is non-starved, the 3:4 hairpin forms in the mRNA. Coordination: As transcription of the trp operon proceeds, the RNA polymerase pauses at the end of sequence 2 until a ribosome begins to translate the leader peptide.

24. Section L: Regulation of transcrip. in Prok.Yang Xu, College of Life Sciences Importance of attenuation 700-fold regulatory effect: Attenuation: The presence of tryptophan gives rise to a 10-fold repression of trp operon transcription through the process of attenuation alone. Trp repressor: Combined with control by the trp repressor (70- fold), this means that tryptophan levels exert a 700-fold regulatory effect on expression from the trp operon. His operon: For example Histidine codons: The His operon has a leader sequence which encodes a peptide with seven successive histidine codons. Only mechanism: The His operon has no repressor-operator regulation, and attenuation forms the only mechanism of feedback control. Other operons: Attenuation occurs in at least six operons that encode enzymes concerned with amino acid bio-synthesis.

25. Section L: Regulation of transcrip. in Prok.Yang Xu, College of Life Sciences L3 Trans. regulation by alternative  factor Sigma factor Heat shock Bateriophage  factor hsp47groel

26. Section L: Regulation of transcrip. in Prok.Yang Xu, College of Life Sciences Regulation patterns and  factors Transcription regulation patterns: By transcriptional repressors: such as the lac repressor; By transcriptional activators: such as the CRP; By different  to direct RNApol binding different promoter: Functions of  factors :  The  ’  core enzyme of RNA polymerase is unable to start transcription at promoter sites.  In order to specifically recognize the consensus -35 and -10 elements of the promoters, it requires the  factor subunit.  This subunit is only required for transcription initiation,  being released from the core enzyme after initiation and before RNA elongation takes place. Features: Many bacteria, including E.coli, produce a set of  factors that recognize different sets of promoters.

27. Section L: Regulation of transcrip. in Prok.Yang Xu, College of Life Sciences

28. Transcriptional regulation by alternative  factor in E. coli for stress condition (an example for the use of different  ) ----- When ＞ 37 ℃ ; (42 ℃ : very soon; 50 ℃ : the only products) Standard  70 ----TTGACA----16-18---------TATAAT------ Responsive Promoter -35 -10 Heat shock  32 ----TTGAA-----13-15---CCCCAT-T---------- ----- When 37 ℃ ; genes expressed in E.coli by RNApol with  70 More then 17 heat-shock proteins are expressed in E.coli through transcription by RNApol using an alternative  32, which have own specific promoter consensus sequence Heat shock gene

29. T4 in E.coli Early gene middle genes late genes host   phage  28 phage  -late Bateriophage  factor Some phages provide new  subunits to the host RNA polymerase with a different promoter specificity and hence to selectively express their own phage genes. This strategy is an effective alternative to the need for the phage to encode its own complete polymerase. This pattern allows its own genes to be transcribed at specific stages during virus infection. proteins

30. Section L: Regulation of transcrip. in Prok.Yang Xu, College of Life Sciences That’s all for Section L