Molecular Biology Course

1. Molecular Biology Course Gene expression controls Transcription often is controlled at the stage of initiation. Transcription is not usually controlled at elongation, but may be controlled at termination to prevent transcription from proceeding past a terminator to the gene(s) beyond. This is the primary control strategy for bacterial gene expression.

2. Molecular Biology Course • In eukaryotic cells, processing of the RNA product can also be regulated at the stages of modification, splicing, transport, or stability. In bacteria, an mRNA is in principle available for translation as soon as it is synthesized, and these stages of control are not available.

3. Molecular Biology Course • Translation may be regulated, usually at the stages of initiation and termination (like transcription). Regulation of initiation is formally analogous to the regulation of transcription: the circuitry can be drawn in similar terms for regulating initiation of transcription on DNA or initiation of translation on RNA. This regulation will not be detailed in this course.

4. Molecular Biology Course Regulationof Transcription in Prokaryotes

5. Regulation of Transcription in Prokaryotes Operon directed regulation • LAC operon • TRP operon s Factor directed regulation

6. Operon Regulation of Transcription in Prokaryotes

7. Regulation of Transcription in Prokaryotes In 1961, Jacob and Monod distinguished between two types of sequences in DNA: sequences that code for trans-acting products; and cis-acting sequences that function exclusively within the DNA. Gene activity is regulatedby the specific interactions of the trans-acting products (usually proteins) with the cis-acting sequences (usually sites in DNA).

8. A gene is a sequence of DNA that codes for a diffusible product. This product may be protein (as in the case of the majority of genes) or may be RNA (as in the case of bgenes that code for tRNA and rRNA etc.). The crucial feature is that the product diffuses away from its site of synthesis to act elsewhere. Any gene product that is free to diffuse to find its target is described as trans-acting.

9. The cis-acting sequence applies to any sequence of DNA that is not converted into any other form, but that functions exclusively as a DNA sequence in situ, affecting only the DNA to which it is physically linked.

10. Regulation of Transcription in Prokaryotes Operon:a unit of prokarytoic gene expression which typically includes: 1. Structural genesfor enzymes in a specific biosynthetic pathway whose expression is co-ordinately controlled 2. Control elements,such asoperator sequence 3. Regulator gene(s)whose products recognize the control elements. Can be encoded by a gene in another operon

11. Control element Structural genes

12. L1 The Lac Operon The operon, the lactose operon, the lac repressor, induction, cAMP receptor protein L2 The TrpOperon The trp operon, the trp repressor, the attenuator, leader RNA structure, the leader peptide, attenuation & its importance L3 Transcriptional regulation by alternative σ Factors Sigma factor, promoter recognition, heat shock, sporulation in B. subtilis, bacteriophage factors

13. Regulation of Transcription in Prokaryotes L1 The Lac Operon • The operon (done) • The lactose operon（乳糖操纵子） • The lac repressor（乳糖抑制蛋白） • Induction （诱导） • cAMP receptor protein （CRP）

14. Overview Lac repressor Transcription blocked The lac operon Inducer (Lactose) High level of transcription Activate the Plac transcription CRP + cAMP (glucose repressed)

15. L1: The LAC operon L1-2 The Lactose Operon E. coli can use lactose as a source of carbon. However, the enzymes required for the use of lactose as a carbon source are only synthesized when lactose is available as the sole carbon source.

16. Lactose operon: a regulatory gene and 3 stuctural genes, and 2 control elements Regulatory gene Structural Genes Cis-acting elements DNA lacI lacZ lacY lacA PlacI Olac Plac m-RNA Protein Transacetylase β-Galactosidase Permease

17. L1: The LAC operon codes for β-galactosidase (半乳糖苷酶) for lactose hydrolysis lacZ encodes a galactoside 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)for lactose metabolism lacA

18. The lacZ, lacY, lacA genes are transcribed from a single (lacZYA) transcription unit under the control of a signal promoter Plac. L1: The LAC operon LacZYA transcription unit contains anoperator site Olac position between bases -5 and +21 at the 3’-end of Plac Binds with the lac repressor

19. Regulation of Transcription in Prokaryotes L1-3 The Lac repressor The repressor is encoded by LacI and active as a tetramer consisting of 4 identical subunits (has a symmetrical structure). It binds to occupies the operator-binding site Olac (28bp, palindromic) and blacks almost all transcription of lacZYA when lack of inducer (such as lactose).

20. Plac

21. L1: The LAC operon The repressor and RNA polymerase can bind simultaneously to the lac promoter and operator sites. The lac repressor actually increases the binding of the polymerase to the lac promoter by two orders of magnitude. Thus,RNA polymerase binds very tightly to Plac but no transcription occur because of the bound repressor

22. L1: The LAC operon L1-4 Induction When lac repressor binds to the inducer (whose presence is dependent on lactose), it changes conformation and cannot bind to Olac site any more. This allows rapid induction of lacZYA transcription.

23. Absence of lactose z y a i p o Active Very low level of lac mRNA Lack of inducer: the lac repressor block all but a very low level of trans-cription of lacZYA . Lactose is present, the low basal level of permease allows its uptake, andβ-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

24. L1: The LAC operon Allolactose causes a change in the conformation of the repressor tetramer , reducing its affinity for the lac operator . The lac operator is removed from the Olac and allows the polymerase to rapidly begin transcription of the lacZYA.

25. Lactose (allolactose) is a native inducer to release RNA transcription elongation from Plac . IPTG, a synthetic inducer, can rapidly simulate transcription of the lac operon structural genes. IPTG is used to induce the expression of the cloned gene from LacZ promoter in many vectors, such as pUC19.

26. L1: The LAC operon Lac promoter MCS (Multiple cloning sites, 多科隆位点） Ampr pUC18 (3 kb) lacZ’ ori Gene X No IPTG, little expression of X gene With IPTG, efficient expression of X gene.

27. L1: The LAC operon L1-5 cAMP receptor protein (CRP) CRP is a transcriptional activator which is activated by binding to cAMP. However, it is only active when cAMP bound, and cAMP is controlled by glucose. CRP activator mediates the global regulation of gene expression from catabolic operons in response to glucose levels.

28. L1: The LAC operon The Plac is a weak promoter, lacking a strong –35 and –10 consensus sequences. High level expression from this promoter requires the activity of the specific activator, CRP.

29. When glucose is present The level of cAMP is low in cell, and CRP exists as a dimer which can’t bind to DNA to regulate transcription. • When glucose is absent The level of cAMP increase and CRP bind to cAMP. The CRP-cAMP complex binds to Plac just upstream from the site for RNA polymerase. Induces a 90°bend in DNA which enhances RNA polymerase binding to the promoter and thus the transcription by 50-fold.

30. CRP-binding site is an inverted repeat.

31. Summary A C B A: RNA polymerase B: lac repressor C: CRP-cAMP

32. The CRP (also called CAP) protein can bind at different sites relative to RNA polymerase. Supp.

33. Regulation of Transcription in Prokaryotes L2 The TrpOperon • The trp operon • the trp repressor • the attenuator • Leader RNA structure • The leader peptide • Attenuation • Importance of attenuation

34. Regulation of Transcription in Prokaryotes L2-1The Trp Operon （色氨酸操纵子 ） Bacillus subtilis uses a different regulation mechanism from what is described here (see the reference of this class).

35. L2: The trp operon 1. The trp operon encodes five structural genes required for tryptophan synthesis. 2. It encodes a signal transcription ( 7kb, polycistron ) downstream of Otrp. 3. These genes are co-ordinately expressed when tryptophan is in short supply in the cell. A B C

36. L2: The trp operon

37. Regulation of Transcription in Prokaryotes L2-2The Trp repressor （色氨酸阻遏物 ）

38. L2: The trp operon 1. Trp repressor is encoded by a separate operon trpR, and specifically interacts with Otrp, a palindrome of 18 bp, and overlaps with the Ptrp sequence between base –21 and +3) 2. The repressor can only bind to the operator Otrp when it is complexed with tryptophan. Therefore, try is a co-repressor and inhibits its own synthesis through end-product inhibition (negative feed-back regulation).

39. L2: The trp operon 3. The repressor reduces transcription initiation by around 70-fold, which is much smaller than the binding of lac repressor. 4. The repressor is a dimer of two subunits which has a structure with a central core and two flexible DNA-reading heads (carboxyl-terminal of each subunit )

40. L2: The trp operon trpR operon trp operon

41. Regulation of Transcription in Prokaryotes L2-3The attenuator （衰减子 ） Repressor does not account for all the regulation: Deletion of a sequence between the operator and trpE gene coding region (attenuator) increase both the basal and the activated (derepressed) levels of transcription.

42. L2: The trp operon 1. Lies at the end of the transcribed leader sequence that precedes the trpE initiator codon. 2. Is a ρ-independent terminator site (GC-rich palindrome) f0llowed by eight successive U residues. 3. Acts as a highly efficient transcription terminator if the hairpin structure is formed, and only a very short transcipt is synthesized.

43. Regulation of Transcription in Prokaryotes L2-4Leader RNA structure （先导RNA的结构 ）

44. L2: The trp operon The leader sequence contains four regions (sequence 1,2,3,4) of complementary sequence that can form different structures Complementary 3:4 termination of transcription Complementary 2:3 Elongation of transcription free leader RNA

45. Regulation of Transcription in Prokaryotes L2-5The leader peptide （先导肽 ） The leader RNA contains an efficient ribosome binding site (RBS) and encodes a 14-amino-acid leader peptide (bases 27-68), Codons 10 and 11 of this peptide encode trp. Thus the availability of trp will affect the translation/ ribosome position, which in turn to regulate transcription termination.

47. Regulation of Transcription in Prokaryotes L2-6Attenuation （衰减作用 ） Transcription and translation in bacteria are coupled. Therefore, synthesis of the leader peptide immediately follows the transcription of leader RNA, and the attenuation is possible

48. L2: The trp operon Transcription of the trp operon During transcription of the RNA from trp operon, the RNA Polymereasepauses at the end of sequence 2(sequences 1 and 2 form a hairpin) until a ribosome began to translate the leader peptide. High trp (attunation) Lack of trp (proceeding through the whole operon )

49. L2: The trp operon High trp Trp is inserted at the trp codons Translate to the end of leader message Ribosome occludesequence 2 Terminate transcription because 3:4 hairpin formed

50. L2: The trp operon Lack of trp Lack of aminoacyl tRNAphe Ribosome pause at trp codons , occluding sequence 1 2:3 hairpin (anti-terminator ) forms Transcription intotrpE and beyond