Chapter 10: Transcription in Bacteria

1. Chapter 10: Transcription in Bacteria

2. Few proteins have had such strong impact on a field as the lac repressor has had in Molecular Biology. Michael Lewis, Comptes Rendus Biologies (2005), 328:521

3. 10.1 Introduction

4. A central event in gene expression is the copying of the sequence of the template strand of a gene into a complementary RNA transcript. • The biochemistry of transcript formation is straightforward. • The regulatory mechanisms that have been developed by bacteria to control transcription are complex and highly variable.

5. 10.2 Mechanism of transcription

6. RNA polymerase is the enzyme that catalyzes RNA synthesis. • Using DNA as a template, RNA polymerase joins, or “polymerizes,” nucleoside triphosphates (NTPs) by phosphodiester bonds from 5' to 3'.

7. In bacteria, transcription and translation are coupled―they occur within a single cellular compartment. • As soon as transcription of the mRNA begins, ribosomes attach and initiate protein synthesis. • The whole process occurs within minutes.

8. Minimal requirements for gene transcription. • Gene promoter • RNA polymerase • Additional factors are required for the regulation of transcription.

9. Bacterial promoter structure • RNA polymerase binds to a region of DNA called a promoter. • Bacterial promoters are not absolutely conserved but they do have a consensus sequence.

10. Conserved sequence: When nucleotide sequences of DNA are aligned with each other, each has exactly the same series of nucleotides in a given region. • Consensus sequence: there is some variation in the sequence but certain nucleotides are present at high frequency.

11. Promoter strength • The relative frequency of transcription initiation. • Related to the affinity of RNA polymerase for the promoter region. • The more closely regions within the promoter resemble the consensus sequences, the greater the strength of the promoter.

12. Structure of bacterial RNA polymerase • Comprised of a core enzyme plus a transcription factor called the sigma factor (). • Together they form the complete, fully functional enzyme complex called the holoenzyme.

13. The core enzyme • The core enzyme catalyzes polymerization. • High affinity for most DNA. • The sequence, structure, and function are evolutionarily conserved from bacteria to humans. • X-ray crystallographic studies revealed a crab claw-like shape.

14. Sigma factor • The sigma () factor decreases the nonspecific binding affinity of the core enzyme. • Binding results in closing of the core enzyme “pincers.” • Primarily involved in recognition of gene promoters.

15. In E. coli the most abundant  factor is 70. • For expression of some genes, bacterial cells use alternative  factors.

16. The sigma factor stimulates tight binding of RNA polymerase to the promoter • Shown over 30 years ago using a nitrocellulose filter binding assay. • The holoenzyme containing  dissociates more slowly from template DNA compared with the core polymerase alone.

17. Initiation of transcription Initiation consists of three stages: • Formation of a closed promoter complex. • Formation of an open promoter complex. • Promoter clearance.

18. Closed promoter complex • RNA polymerase holoenzyme binds to the promoter at nucleotide positions 35 and 10. • The DNA remains double-stranded. • The complex is reversible.

19. Open promoter complex • ~18 bp around the transcription start site are melted to expose the template strand DNA. • AT rich promoters require less energy to melt. • Transcription is aided by negative supercoiling of the promoter region of some genes. • The open complex is generally irreversible.

20. Transcription is initiated in the presence of NTPs. • No primer is required for initiation by RNA polymerase.

21. Promoter clearance Older “classic” model •  factor release. Current model •  factor does not completely dissociate; some domains are displaced. • The displaced domains allow the nascent RNA to emerge from the RNA exit channel.

22. Elongation • After about 9-12 nt of RNA have been synthesized, the initiation complex enters the elongation stage.

23. Direction of transcription around the E. coli chromosome • Of the 50 operons or genes whose transcription direction is known, 27 are transcribed clockwise and 23 in the counterclockwise direction around the circle, using the opposite strand as a template. • Only one strand of a given operon’s DNA is used as a template for transcription.

24. The origin and terminus of replication divide the genome into oppositely replicated halves or “replichores.” • Most operons or genes are transcribed in the direction of replication. • This may lead to fewer collisions of DNA and RNA polymerase and less topological strain from opposing supercoils.

25. As RNA polymerase moves during elongation, it holds the DNA strands apart, forming a transcription “bubble.” • The moving polymerase protects a “footprint” of ~30 bp along the DNA against nuclease digestion.

26. One strand of DNA acts as the template for RNA synthesis by complementary base pairing. • The catalytic site has both a substrate-binding and a product-binding site. • Transcription always proceeds in the 5′→3′ direction.

27. Completion of the single nucleotide addition cycle. • Shift of the active site of the RNA polymerase by one position along the template DNA.

28. Which moves – the RNA polymerase or the DNA? Two models • Model 1: RNA polymerase moves along and the DNA rotates. • This is the more widely accepted model. • Model 2:RNA polymerase remains stationary, and the DNA moves along and rotates.

29. The overall process of transcription has a significant local effect on DNA structure • The DNA ahead of the RNA polymerase is wound more tightly; positive supercoils form. • Behind the polymerase, DNA becomes less tightly wound; negative supercoils form. • Topoisomerase I and gyrase (bacterial topoisomerase II) resolve this supercoiling and restore the DNA to its relaxed form.

30. RNA polymerase is a molecular motor • RNA polymerase rotates the DNA. • Tracks the DNA helix over thousands of base pairs, producing measurable torque. • A real-time optical microscopy in vitro assay was used to catch RNA polymerase in the act of transcribing.

31. Proofreading Proofreading by RNA polymerase • Backtracks 3′→5′ • Pauses • Nucleolytic cleavage

32. Termination of transcription In most bacteria, there are two types of terminators: • Rho-independent • Rho-dependent

33. Rho-independent termination • Terminator is characterized by an inverted repeat consensus sequence. • Formation of a stem-loop in the exit channel. • Less stable U-A hybrid helix. • Polymerase pauses, resulting in transcript release.

34. Rho-dependent termination • Terminator is an inverted repeat with no simple consensus sequence. • Controlled by the ability of the Rho protein to gain access to the mRNA. • Because ribosomes translate mRNA at the same rate as the mRNA is transcribed, Rho is prevented from loading onto the newly formed RNA until the end of a gene or operon.

35. Rho binds specifically to a C-rich site called a Rho utilization (rut) site. • ATP-dependent polymerase “chasing.” • Polymerase pauses at the terminator stem-loop structure, Rho catches up and unwinds the DNA-RNA hybrid. • Transcript release.

36. 10.3 Insights into gene regulation from the lactose (lac) operon

37. The 1959 operon model of Jacob and Monod • Novel concept of regulatory genes that code for products that control other genes. • Model predicted the existence of an unstable RNA as an intermediate in protein synthesis.

38. The Jacob-Monod operon model of gene regulation • Model arose from experimental observations in bacteria and phages. • Study of how phage lambda () can be induced to switch from lysogenic to lytic state. • Study of how the enzyme -galactosidase is produced in bacterial cells only when bacteria need this enzyme to use the sugar lactose

39. The lac operon provides an example of negative control of the enzymes involved in lactose metabolism. • The lac operon is also regulated by positive control under certain environmental conditions.

40. Characterization of the Lac repressor • Jacob-Monod model also proposed the existence of a repressor protein. • Gilbert and Müller-Hill isolated the Lac repressor and demonstrated that it binds operator DNA.

41. Lactose (lac) operon regulation • In bacteria, genes are organized into operons. • An operon is a unit of bacterial gene expression and regulation, including structural genes and control elements in DNA recognized by regulatory gene product(s). • Transcribed from a single promoter to produce a single primary transcript of polycistronic mRNA.

42. In eukaryotes, genes are not typically organized into operons. • Exception • ~15% of genes in Caenorhabditis elegans are grouped into operons. • But, each C. elegans pre-mRNA is processed into a separate mRNA for each gene rather than being translated as a unit.

43. Bacteria need to respond swiftly to changes in their environment, switching from metabolizing one substrate to another quickly and efficiently. • Inductionis the synthesis of enzymes in response to the appearance of a specific substrate. • When provided with a mixture of sugars, bacteria use glucose first.

44. Cis-acting DNA sequence Trans-acting factor gene Gene coding region Transcription Translation Unifying theme in gene transcription • A regulatory protein (trans-acting factor) binds to a particular sequence of DNA (cis-acting factor)

45. Lac operon induction • The lac operon consists of three structural genes, lacZ, lacY, and lacA. • lacZ encodes -galactosidase, an enzyme which cleaves lactose into galactose and glucose. • lacY encodes a lactose permease, part of the transport system to bring lactose into the cell. • lacA encodes a transacetylase that rids the cell of toxic thioglactosides that get taken up by the permease.

46. In the absence of lactose, the Lac repressor binds the operator and excludes RNA polymerase. • In the presence of lactose the lac operon is induced. • Recruitment of RNA polymerase requires formation of a complex of the cAMP-bound activator protein CAP, polymerase, and DNA.

47. Allolactose – the real inducer • The real inducer of the lac operon is an alternative form of lactose called allolactose. • When -galactosidase cleaves lactose, it rearranges a small fraction of the lactose to allolactose.

48. Basal transcription of the lac operon • The basal level of transcription is determined by the frequency with which RNA polymerase spontaneously binds the promoter and initiates transcription.

49. The lac operon is transcribed if and only if lactose is present in the medium. • But, this signal is almost entirely overridden by the simultaneous presence of glucose.

50. Glucose exerts its effect, in part, by decreasing synthesis of cAMP which is required for the activator CAP to bind DNA. • More importantly, however, glucose inactivates the lactose permease. • Without cooperative binding of CAP, RNA polymerase transcribes the lac genes at low level.