Lecture 5-6

Patterns of DNA Synthesis

• A replicon is a DNA molecule that replicates from a single origin.
• Bacterial DNA is typically circular and replicates bidirectionally from one origin.
• The replication fork is where DNA unwinds during replication.
• Most bacteria have one replicon, but some, like Vibrio cholerae, have multiple replicons.

DNA Replication & Transcription Summary

• DnaA is an essential initiation factor protein.
• Replication begins when DnaA binds to DnaA box sequences, causing DNA to wrap around and melt the AT-rich region.

Replication Fork Events (E. coli)

• DNA polymerase synthesizes new DNA in the 5’ to 3’ direction.
• DNA helicase, discovered in E. coli in 1976, is an ATP-dependent enzyme that separates parental strands and unwinds DNA at the origin of replication.
• The leading strand undergoes continuous synthesis.
• The lagging strand is synthesized in Okazaki fragments, requiring new primers for each fragment.
• CLC (Clamp Loader Complex) and SSB (Single-Strand Binding Protein) are key proteins in this process.

Linking Okazaki Fragments

• DNA ligase forms phosphodiester bonds between Okazaki fragments.

DNA Proofreading

• DNA polymerases use exonuclease activity at the 3’ end to remove mismatched bases, though this process is not 100% efficient.
• Most RNA viruses do not have proofreading mechanisms.

Transcription

• Transcription and translation occur in the cytoplasm.
• RNA is synthesized from a DNA template, creating a complementary RNA sequence.
• mRNA carries genetic instructions for proteins.
• tRNA transfers amino acids.
• rRNA is a structural component of ribosomes.
• Regulatory RNA includes RNAi, miRNA, and siRNA, which control gene expression.
• tRNA and rRNA genes are often transcribed as single precursor molecules.
• Some tRNA genes encode multiple tRNA types.
• Spacer regions are removed post-transcription using ribozymes.

Protein Synthesis Summary

• The template strand (3’ → 5’) directs RNA synthesis, and mRNA is synthesized 5’ → 3’.
• The non-template strand matches mRNA (except T is replaced by U).
• The promoter is the RNA polymerase binding site at the start of a gene.
• The sigma (σ) factor helps RNA polymerase bind to the promoter.

Transcription Termination

• The leader sequence is transcribed but not translated.
• The Shine-Dalgarno sequence is a ribosome binding site essential for translation initiation.

The Genetic Code

• A codon is a 3-base genetic code unit that specifies an amino acid.
• The anticodon (tRNA) is complementary to the codon.
• The start codon is usually AUG (also GUG, UUG, CUG), all of which are translated as Met.
• There are 61 sense codons that encode amino acids.
• Stop codons are UAG, UAA, and UGA, which do not encode amino acids.
• Code degeneracy means that some amino acids have multiple codons.
• The Poly-U Experiment used radiolabeled amino acids to determine that UUU codes for Phenylalanine.
• There was no start codon, but high Mg²⁺ levels allowed translation.

tRNA & Amino Acid Activation

• The anticodon binds to the codon on mRNA.
• The 3’ end of tRNA binds to the amino acid.
• Aminoacyl-tRNA synthetases attach amino acids to tRNA, one for each amino acid.

Ribosomal RNA & Translation

• 16S rRNA binds the Shine-Dalgarno sequence and helps initiate translation and recruit aminoacyl-tRNA.
• 23S rRNA acts as a ribozyme, catalyzing peptide bond formation.

Translation Initiation

• Peptidyl transferase (23S rRNA ribozyme) catalyzes bond formation in the transpeptidation reaction.
• The A-site amino acid reacts with the P-site tRNA, transferring the peptide chain.

Regulation of Bacterial Cellular Processes

• Regulatory mechanisms include regulatory proteins, attenuators, and riboswitches.
• Regulatory proteins bind to DNA to enhance or inhibit transcription initiation.
• Attenuators and riboswitches bind to mRNA to enhance or inhibit transcription elongation.
• The leader sequence, also known as the Shine-Dalgarno (SD) region, is the translation initiation site (ribosomal binding site).
• Polycistronic gene clusters contain one or more genes regulated together.

Regulation of Transcription Initiation

• Regulates the synthesis of necessary enzymes or the replacement of degraded ones.
• Repressible (constitutive) genes, or housekeeping genes, are continuously expressed (e.g., DNA polymerase).
• Inducible genes are turned on/off as needed in response to environmental changes, such as β-Galactosidase.
• β-Galactosidase breaks lactose into galactose + glucose.

Control of Transcription Initiation by Regulatory Proteins

• Regulatory proteins inhibit transcription through negative control and promote transcription through positive control.
• Under negative control (inducible genes), a repressor inhibits transcription unless an inducer removes it (e.g., catabolic pathways).
• Under positive control (inducible genes), an activator promotes transcription in the presence of an inducer (e.g., catabolic pathways).
• Under negative control (repressible genes), a repressor blocks transcription when a corepressor is present (e.g., anabolic pathways).
• Under positive control (repressible genes), an activator promotes transcription unless an inhibitor prevents it (e.g., anabolic pathways).
• Catabolic pathways are usually inducible.
• Anabolic pathways are usually repressible.
• Catabolic pathways activate only when the substrate is available and there is no preferred energy source.
• Anabolic pathways shut down when enough product has been synthesized to conserve energy and resources.

Negative Control of Transcription Initiation

• A repressor binds to the operator, inhibiting transcription.
• Repressor proteins exist in active/inactive forms.
• Inducers (substrates) bind to the repressor to inactivate it, allowing transcription to proceed.
• Corepressors (products) bind to the repressor to activate it, blocking transcription.

Positive Control of Transcription Initiation

• An activator binds to the regulatory region, promoting transcription.
• Activators require an inducer or inhibitor to function.

The lac Operon of E. coli

• The lac operon of E. coli is under both positive and negative control.
• It is an inducible catabolic pathway that breaks lactose into glucose + galactose.
• It is only needed when glucose is absent.

Regulation of the lac Operon

• In negative control (repression), the lacI repressor binds to the operator, blocking transcription.
• Lactose (the inducer) binds to the repressor, inactivating it, which allows transcription to occur.
• In positive control (activation), the Catabolite Activator Protein (CAP) binds to the CAP site, enhancing transcription.
• This happens only when glucose is absent.
• Lactose permease uses an H+ gradient to transport lactose into the cell.
• β-Galactosidase (β-Gal) catalyzes the hydrolysis of lactose into galactose + glucose.
• A side reaction of β-Galactosidase occasionally forms allolactose.
• Allolactose signals the presence of lactose and functions as the inducer for the lac operon.

lacI Repressor

• The lacI repressor forms tetramers that bind to three operator sites (O1, O2, O3).
• It bends DNA, preventing RNA polymerase from binding to the promoter.
• Allolactose binds to the lacI repressor, inactivating it, which allows transcription of the lac operon genes.

Catabolite Activator Protein (CAP) and cAMP Regulation

• CAP exists in two forms: an active form bound to cyclic AMP (cAMP) and an inactive form when not bound to cAMP.
• cAMP levels are controlled by adenylate cyclase, which converts ATP into cAMP + PPi.
• Glucose inhibits adenylate cyclase, reducing cAMP levels.
• Low glucose leads to high cAMP, which binds to CAP, activating the lac operon.
• High glucose leads to low cAMP, making CAP inactive and keeping the lac operon off.

Diauxic Growth

• Diauxic growth is a biphasic growth pattern where bacteria prefer one carbon source over another.
• The preferred substrate (e.g., glucose) is used first.
• A lag phase occurs when the preferred substrate is exhausted.
• Bacteria switch to the secondary substrate (e.g., lactose), resuming growth at a different rate.
• Different slopes on a growth graph represent different growth rates for each carbon source.

Regulation of the Arabinose and Tryptophan Operons

• The arabinose (ara) operon is a catabolic pathway that breaks down arabinose.
• Transcriptional control is managed by a dual-function protein that acts as both an activator and a repressor.
• Regulation depends on the presence of arabinose for activation and the presence of glucose for repression.
• The tryptophan (trp) operon is an anabolic pathway that synthesizes tryptophan.
• It consists of five structural genes that code for enzymes required for tryptophan synthesis.
• Regulation is under negative transcriptional control of repressible genes.
• The operon is active only when tryptophan is absent.
• When tryptophan is present, the operon is repressed to prevent unnecessary synthesis.

Attenuation of the trp Operon

• Attenuation is a second form of negative feedback regulation that allows fine-tuning of tryptophan synthesis by linking transcription and translation.
• Repression responds to tryptophan concentration (TrpR repressor).
• Attenuation responds to tryptophan tRNA concentration.
• The TrpR repressor decreases transcription 70-fold.
• Attenuation further decreases transcription 10-fold.
• Total repression results in an approximately 700-fold reduction in trp operon activity.

Additional Regulatory Mechanisms

• Riboswitches are a form of transcription or translation attenuation.
• The leader sequence (riboswitch) folds into different structures, determining if transcription or translation continues or terminates.
• The structure is altered in response to an effector molecule.
• Regulation at the level of translation regulates mRNA translation instead of transcription.
• For example, vitamin B1 and B12 transporters are repressed if sufficient molecules are detected in the cell.

Two-Component Regulatory Systems

• Many genes and operons are regulated in response to environmental conditions.
• The Two-Component Signal System links extracellular conditions to gene expression regulation, allowing bacteria to respond quickly to environmental changes.
• EnvZ is a membrane-bound sensor kinase that detects osmolarity changes in the environment.
• High osmolarity causes EnvZ to phosphorylate OmpR, activating it.
• OmpR is a cytoplasmic response regulator that regulates the transcription of porin proteins based on osmolarity.
• Activated OmpR (OmpR-P) controls OmpC and OmpF expression.
• OmpC is a small porin with a smaller pore size, dominant in high osmolarity environments (e.g., the intestinal tract with high salt/sugar concentrations).
• It reduces diffusion into the cell, preventing excessive uptake of harmful substances.
• OmpF is a large porin with a larger pore size, dominant in low osmolarity environments (e.g., freshwater).
• Its 10-fold faster diffusion rate helps maximize nutrient uptake when resources are scarce.

Regulation of OmpF and OmpC by OmpR-P

• In low osmolarity, there are low levels of activated OmpR-P, which allows OmpF transcription to proceed and inhibits OmpC.
• In high osmolarity, there are high levels of activated OmpR-P, which represses OmpF and activates OmpC transcription.
• OmpR-P has a higher affinity for the OmpF promoter than for OmpC.
• Within OmpF, OmpR-P binds strongly to F1, F2, and F3 sites but weakly to F4.
• micF Gene: Activated by OmpR-P in high osmolarity.
• micF RNA (antisense RNA) binds to OmpF mRNA, preventing its translation.
• Ensures that even if OmpF mRNA is transcribed, it is not translated into protein, reinforcing OmpF repression in high osmolarity environments.

Summary of Regulation Mechanism

• Low osmolarity results in low OmpR-P levels, inhibited OmpC expression, activated OmpF expression, and no micF RNA expression.
• High osmolarity results in high OmpR-P levels, activated OmpC expression, repressed OmpF expression, and micF RNA binding to OmpF mRNA to prevent translation.