Lactose Metabolism and Lac Operon in E. coli

Lactose Metabolism and the Lac Operon of E. coli

• Lactose is a major carbohydrate in milk, metabolized by E. coli bacteria in the mammalian gut.
• Lactose, to be used as an energy source, must be transported into the E. coli cell by permease.
• Permease actively transports lactose into the cell.
• Beta-galactosidase breaks lactose into galactose and glucose.
• Beta-galactosidase can also convert lactose to allolactose.
• Allolactose plays a crucial role in regulating lactose metabolism.
• Thiogalactoside transacetylase is another enzyme produced by the lac operon, but its function in lactose metabolism is not yet known.

Regulation of the lac Operon

• The lac operon is a negative inducible operon.
• The enzymes beta-galactosidase, permease, and transacetylase are encoded by adjacent genes in the lac operon (lacZ, lacY, and lacA).
• These enzymes share a common promoter (lacP).
• If lactose is present in the medium and glucose is absent, synthesis of the three proteins increases a thousand-fold within 2-3 minutes, coordinated induction.
• The lacI gene codes for a repressor
• The repressor binds to laco (the operator) which overlaps the 3' end of the promoter and the 5' end of lacZ.
• When the repressor binds to the operator, RNA polymerase is blocked, and transcription is prevented.
• When lactose is present, some is converted to allolactose, which binds to the repressor.
• This binding inactivates the repressor, releasing it from DNA.
• RNA polymerase is no longer blocked, transcription of lacZ, lacY, and lacA takes place, and lac proteins are produced.

The trp Operon of E. coli

• The trp operon controls the biosynthesis of the amino acid tryptophan.
• It is a negative repressible operon.
• Transcription is normally "on" and must be repressed.
• When tryptophan levels are low, transcription occurs.
• When tryptophan levels are high, tryptophan binds to the repressor, making it active.
• The trp repressor binds to the operator, stopping RNA polymerase from transcribing the genes.

Regulation of Transcription Through Attenuation

• Some operons, like the trp operon, regulate transcription through attenuation, a process that affects the continuation of transcription rather than its initiation.
• Transcription begins at the start site, but termination takes place prematurely, before RNA polymerase reaches the structural genes.

RNA Molecules Controlling Gene Expression

• Some small RNA molecules, called antisense RNA, are complementary to particular sequences on mRNA.
• Antisense RNA controls gene expression by binding and inhibiting translation.
• The ompF gene encodes an outer-membrane protein and functions in the passive diffusion of small polar molecules.
• When osmolarity increases, the cell depresses OmpF protein production, regulating osmolarity.

Riboswitches

• mRNA molecules contain regulatory sequences called riboswitches.
• They control gene expression by influencing the formation of secondary structures in the mRNA.
• Riboswitches are often found in the 5' UTR of mRNA molecules and fold into compact secondary structures.
• Some can cause premature termination of transcription or prevent translation initiation, depending on the presence of a specific small molecule.

Gene Regulation in Eukaryotes

• Eukaryotic gene regulation is more complex than bacterial gene regulation.
• Genes are not usually organized into operons.
• Chromatin structure affects transcription, as DNA must unwind for transcription to occur.
• Changes in chromatin structure, such as histone modifications and chromatin remodeling, alter gene expression.
• DNA methylation can affect chromatin structure and gene expression.
• Transcription factors and transcriptional regulator proteins control gene initiation.
• Activators stimulate transcription, while repressors inhibit it.
• Enhancers are regulatory sequences that can stimulate transcription from a distance.
• Insulators limit the effects of enhancers and prevent unwanted interactions.

mRNA Editing

• mRNA editing is a process where sequences of mRNA change after transcription.
• A significant example is the production of apolipoprotein B100 and apolipoprotein B48.

Alternative Splicing

• An example of this is the T-antigen gene of the SV40 virus, where different proteins (large T and small t antigens,) can be produced from the same gene depending on which splice sites are used.

RNA Degradation

• The amount of protein synthesized depends on the amount of mRNA available for translation.
• The amount of mRNA depends on the rates of mRNA synthesis and mRNA degradation.
• Various mRNA degradation mechanisms exist.

RNA Interference

• RNA interference (RNAi) is a process that controls gene expression.
• It employs small interfering RNAs (siRNAs) and microRNAs (miRNAs), which pair with target mRNA molecules, leading to mRNA cleavage, translation inhibition, transcriptional silencing, and mRNA degradation.

Post-translational Processing of proteins

• Polypeptides emerging from ribosomes are often inactive and need post-translational modifications.
• These modifications can include folding, cleavage proteolysis, chemical modifications, and modification of protein intein splicing to activate or alter the protein's function and activity.