Transcription(I)

1. Transcription(I) 王之仰

2. Transcription is a complex process involving many layers of regulation. • Repressor and activator proteins recognize and bind to specific regions of DNA to control the transcription of a nearby gene. • In E. coli, about half of genes are

4. clustered into operons, each of which encodes enzymes involved in a particular metabolic pathway or proteins that interact to form one multisubunit protein. • All the genes within an operon are coordinately regulated.

5. Transcription of operons is controlled by an interplay between RNA polymerase and specific repressor and activator proteins. • E. coli RNA polymerase must be associated with one of a small number of σ(sigma)

6. factors. • The most common one in eubacterial cells is σ70. • σ70 binds to RNA polymerase and to promoter DNA sequences, bringing the RNA polymerase to a promoter. • σ70 recognizes and binds to both

7. a six-base pair sequence centered at -10 and a seven-base pair sequence centered at -35 from the +1 transcription start. • E. coli RNA polymerase binds to the promoter region DNA from -50 to -20 through interactions with DNA.

8. E. coli RNA polymerase binds to the promoter region DNA from -50 to -20 through interactions with DNA that do not depend on the sequence. • σ70 can assist the RNA polymerase in separating the DNA strands at the transcription start site and inserting the coding

9. strand into the active site of the polymerase so that transcription starts at +1. • The optimal σ70-RNA polymerase promoter sequence, determined as the consensus sequence of multiple strong promoters, is TTGACAT (-35)-15-17 bp- TATAAT (-10).

10. σ70 acts as an initiation factor required for transcription initiation but not for RNA-strand elongation once initiation has taken place. • When E. coli is in an environment that lacks lactose, synthesis of lac mRNA is repressed so that cellular energy is not wasted synthesizing enzymes the cell cannot use.

11. E. coli preferentially metabolize glucose and lactose is metabolized at a high rate only when lactose is present and glucose is largely depleted from the medium. • Transcription of the lac operon under different conditions is controlled by lac repressor and catabolite activator protein (CAP). Each of which binds to a

12. specific DNA sequence in the lac transcription-control region. • For transcription of the lac operon to begin, the σ70 subunit must bind to the lac promoter at the -35 and -10 promoter sequences; when no lactose is present, the lac repressor binds to a sequence called the lac operator, which overlaps the transcription start site.

13. When lactose is present, it binds to specific binding sites in each subunit of the tetrameric lac repressor, causing a conformational changes in the protein that makes it dissociate from the lac operator. • Once glucose is depleted from the media and the intracellular glucose concentration falls, E. coli respond by

14. synthesizing cyclic AMP. • As the concentration of cAMP increases, it binds to a site in each subunit of the dimeric CAP protein allowing the protein to bind to the CAP site in the lac transcription-control region. • The bound CAP-cAMP complex interacts with the polymerase bound to the promoter, stimulating the rate of

16. transcription initiation. • The tetrameric lac repressor actually binds to two sites simultaneously, one at the primary operator (lacO1) that overlaps the region of DNA bound by RNA polymerase at the promoter and at one of two secondary operators centered at +412 (lacO2) and -82(lacO3).

17. Simultaneous binding of the tetrameric lac repressor to the primary lac operator O1 and one of the two secondary operators is possible because DNA is quite flexible. • These secondary operators function to increase the local cencentration of lac repressor in the micro-vicinity of the primary operator where repressor

18. repressor binding blocks RNA polymerase binding. • Since the equilibrium of binding reactions depends on the concentration of the binding partners, the resulting increased local concentration of lac repressor in the vicinity of O1 increases repressor binding to O1. • Mutation of only O2 or O3 reduces

20. repression twofold, indicating that either one of these secondary operators provides most of the stimulation of repression. • Promoters that support a high rate of transcription initiation have -10 and -35 sequences similar to the ideal promoter shown previously and are called strong promoter.

21. Those that support a low rate of transcription initiation differ from this ideal sequence and are called weak promoters. • Transcription of most E. coli is involved in a specific repressor that binds to the operator region of a gene or operon, thereby blocking transcription initiation. • Specific activator proteins, such as CAP

22. in the lac operon, control transcription of a subset of bacterial genes that have binding sites for the activator. • Specific activator proteins, such as CAP, and others bind to DNA together with RNA polymerase, stimulating transcription from a specific promoter. • An activator can be modulated by cAMP or by phosphorylation.

23. Most E. coli promoters interact with σ70 RNA polymerase. • Alternative σ-factors are required for the transcription of sets of genes with related functions involved in the response to heat shock or nutrient deprivation, motility, or sporulation in G(-) bacteria.

25. Streptomyces encodes 63 σ-factors ; most are structurally and functionally related to σ70; but one class is unrelated, represented in E. coli by σ54. • Transcription of genes by RNA polymerases containing σ54 is regulated solely by activators whose binding sites in DNA, referred to as enhancers, located 80-160 bps upstream

26. from the start site. • The best-characterized σ54-activator-the NtrC protein-stimulates transcription of the glnA gene; glnA encodes the enzyme glutamine synthetase, which synthesizes the amino acid glutamine from glutamic acid and ammonia. • The σ54-RNA polymerase binds to the glnA promoter but does not melt the

27. DNA strands and initiate transcription until it is activated by NtrC, a dimeric protein; NtrC is regulated by a protein kinase called NtrB. • In response to low levels of glutamine, NtrB phosphorylates NtrC, which then binds to an enhancer upstream of the glnA promoter. • Enhancer-bound phosphorylated NtrC

28. then stimulates the σ54-polymerase bound at the promoter to separate the DNA strands and initiate transcription. • Electron microscopy studies have shown that phosphorylated NtrC bound at enhancers and σ54-polymerase bound at the promoter directly interact, forming a loop in the DNA between the binding sites.

29. NtrC has ATPase activity, and ATP hydrolysis is required for activation of bound σ54-polymerase by phosphorylated NtrC; ATP hydrolysis supplies the energy required for melting the DNA strands. In contrast, the σ70-polymerase does not require ATP hydrolysis to separate the strands at a start site.

32. Control of the E. coli glnA gene depends on two proteins, NtrC and NtrB- two-component regulatory systems control many responses of bacteria to changes in their environment. • The E. coli proteins, PhoR and PhoB, regulate transcription in response to the concentration of free phosphate. PhoR is a transmembrane protein, located in

33. the inner membrane, whose periplasmic domain binds phosphate with moderate affinity and whose cytosolic domain has protein kinase activity; PhoB is a cytosolic protein. • Large protein pores in the E.coli outer membrane allow ions to diffuse freely between the external environment and the periplasmic space. When the

34. phosphate concentration in the environment falls, it also falls in the periplasmic space, causing phosphate to dissociate from the PhoR periplasmic domain. This causes a conformational change in the PhoR cytoplasmic domain that activates its protein kinase activity. • The activated PhoR initially transfers a γ-phosphate from ATP to a histidine (H)

35. side chain in the PhoR kinase domain. • The same phosphate is then transferred to a specific aspartic acid (D) side chain in PhoB, converting PhoB from an inactive to an active transcriptional activator. Phosphorylated, active PhoB then induces transcription from several genes that help the cell cope with low phosphate conditions.

36. Other bacterial responses are regulated by two proteins; one protein, called a sensor, contains a transmitter domain homologous to the PhoR protein kinase domain. • The transmitter domain of the sensor protein is regulated by a second unique protein domain that senses environmental changes.

37. The second protein, called a response regulator, contains a receiver domain homologous to the region of PhoB that is phosphorylated by activated PhoR. • The receiver domain of the response regulator is associated with a second domain that determines the protein’s function. • The activity of this second functional

38. domain is regulated by phosphorylation of the receiver domain; The transmitter domain of a specific sensor protein will phosphorylate only the receiver domains of specific response regulators. • NtrB and NtrC function as sensor and response regulator proteins in the two-component regulatory system that controls transcription of glnA.