Prokaryotic Transcription and DNase Footprinting

Prokaryotic Transcription

• Transcription is DNA-templated RNA synthesis
• Primary transcript is the immediate product of transcription in prokaryotes
• Pre-mRNA is processed to become mRNA in eukaryotes
• Ribonucleotides are added in a 5' to 3' direction, forming phosphodiester bonds
• RNA polymerase catalyzes RNA synthesis
• E. coli RNA polymerase has multiple subunits (σ, β, β', α, ω)
• σ subunit separates from core enzyme to form holoenzyme
• σ70 is a common σ subunit in E. coli
• Promoters are RNA polymerase binding sites
• Sigma factors and promoters can be studied using a filter binding assay
• Core enzyme and holoenzyme bind DNA differently; temperature affects binding
• Promoters have specific regions (e.g., -10 and -35) crucial for holoenzyme recognition.
• -10 region (Pribnow box) consensus sequence is TATAAT
• -35 region consensus sequence is TTGACA
• Mutations affecting promoter strength are called up or down mutations

DNase Footprinting

• Used to identify protein-DNA interactions
• Labeling, protein binding, then DNase treatment reveals binding sites
• Different sigma factors recognize different consensus sequences in core promoters

Transcription Stages

• Initiation: RNA polymerase binds to promoter, forms closed and open complexes, the σ subunit dissociates
• Elongation: RNA polymerase synthesizes RNA in a 5' to 3' direction
• Termination: Two types: Intrinsic (RNA polymerase pauses, forms hairpin loop) and Rho-dependent (Rho protein detaches RNA from polymerase)

Operons

• Operons group genes for related functions together for coordinated regulation
• The lac operon is an example of coordinated gene expression
• Diauxic growth is metabolizing preferred sugars first, then switching to second sugars
• Lac operon regulation depends on lactose (inducer) presence and levels of glucose

Lac Repressor

• Lac repressor is a homotetrameric protein
• Repressor has regulatory and DNA-binding domains
• Binds to the operator sequence when lactose is absent.
• Lactose, is converted to allolactose, which causes repressor to release, enabling transcription

Positive Control

• Cyclic AMP (cAMP) is a small metabolite related to low glucose
• cAMP receptor protein (CRP) binds cAMP
• CRP-cAMP complex binds to DNA and recruits RNA polymerase
• CRP-cAMP complex helps RNA polymerase bind promoters

Arabinose Operon

• Arabinose operon genes are coordinately regulated around an ara promoter
• AraC is a negative AND positive regulator.
• In absence of arabinose, AraC binds to DNA, looping it to block transcription

Galactose Operon

• Galactose operon has negative control, unlike Lac, which prevents the transition from closed to open complex preventing elongation
• Galactose is an alternative energy source for E. coli

Tryptophan Operon

• The trp operon controls the synthesis of tryptophan
• Operon expression is regulated negatively by tryptophan; when tryptophan is abundant, tryptophan binds repressor activating it (co-repressor).
• Attenuation of trp operon transcription can occur before polymerase reaches the tryptophan synthesis genes

Global Changes in Bacterial Transcription

• Sigma factors are crucial for RNA polymerase specificity
• Time-dependent programming occurs via switching sigma factors
• Sporulation and phage infection alter sigma factors for regulated gene expression
• Several specific sigma factors are needed for the sporulation process in Bacillus subtilis, enabling the expression of genes in certain phases of the process

Riboswitches

• Riboswitches are RNA molecules with small-molecule-binding elements that influence expression depending on the presence of ligand

Heat Shock Response

• Heat-shock response is rapid
• This is caused by the sigma factor 32 (RpoH) being free to bind, enabling transcription of heat shock proteins