In prokaryotic cells, RNA polymerase binds to the promoter, but it can only find and recognize the promoter when attached to a protein called the sigma (σ) factor. Together, the core RNA polymerase enzyme and the sigma factor form what’s known as the holoenzyme, and this complete complex is what actually lands on promoter DNA to start transcription. Beyond the holoenzyme, regulatory proteins like activators and repressors also bind at or near the promoter to control whether transcription happens.
RNA Polymerase and the Sigma Factor
The core RNA polymerase in prokaryotes is a large protein complex, roughly 400 kilodaltons, made up of five subunits: two alpha (α) subunits, one beta (β), one beta-prime (β’), and one omega (ω). This core enzyme can read DNA and build RNA, but on its own it can’t locate a promoter. It would just bind DNA randomly.
The sigma factor solves this problem. When sigma attaches to the core enzyme, it creates the holoenzyme, which can scan along DNA and specifically recognize promoter sequences. Sigma is the subunit that makes direct, sequence-specific contact with promoter DNA. Once transcription begins and the RNA polymerase starts moving along the gene, the sigma factor releases from the complex, leaving the core enzyme to finish the job.
What the Sigma Factor Recognizes
Prokaryotic promoters contain two critical DNA sequences that the sigma factor reads. The first is the -10 element (also called the Pribnow box), located about 10 base pairs upstream of where transcription starts, with a consensus sequence of TATAAT. The second is the -35 element, located about 35 base pairs upstream, with a consensus sequence of TTGACA. The closer a promoter’s actual sequence matches these consensus sequences, the stronger the promoter tends to be.
Different parts of the sigma factor handle each element. A region called conserved region 4 acts as a helix-turn-helix DNA-binding module that recognizes the -35 element as double-stranded DNA. Conserved region 2 recognizes the -10 element and also plays a key role in the next critical step: prying open the two DNA strands. Aromatic amino acids in region 2 stack against the bases of the separated non-template strand, essentially providing a protein scaffold that holds the “bubble” open. This strand separation exposes the template strand so RNA polymerase can read it, creating what’s called the open complex. The holoenzyme typically unwinds 12 to 14 base pairs of DNA during this process.
The UP Element and Alpha Subunit
Some promoters have an additional sequence called the UP element, located upstream of the -35 region (roughly positions -40 to -60). The UP element is AT-rich and is recognized not by sigma but by the C-terminal domain of the alpha subunit (α-CTD). This interaction uses a DNA-binding architecture unlike any other known protein-DNA binding mode. When the α-CTD grabs onto the UP element, it boosts RNA polymerase’s affinity for the promoter significantly, making these promoters among the strongest in the cell. The ribosomal RNA promoters in E. coli, which need to produce enormous quantities of ribosomes in growing cells, are a classic example.
Closed Complex to Open Complex
Promoter binding happens in stages. First, the holoenzyme recognizes the -35 and -10 elements and sits down on double-stranded DNA. This is the closed complex. The DNA strands are still paired, and no transcription can occur yet. Next, the sigma factor drives strand separation around the -10 region, flipping the non-template strand out and threading the template strand toward the enzyme’s active site. A loop within sigma called the σ3.2 finger extends into the RNA polymerase’s active-site channel and helps guide the template strand into position. The result is the open complex, where about 12 to 14 base pairs are unwound and the enzyme is ready to begin synthesizing RNA.
This transition from closed to open complex is temperature-dependent. Higher temperatures make it easier for the DNA strands to separate, which is why some experiments at low temperatures can trap the enzyme in the closed complex before it melts the DNA open.
Different Sigma Factors for Different Conditions
Bacteria don’t rely on a single sigma factor. E. coli, for example, produces several, each recognizing different promoter sequences and activating different sets of genes. The primary sigma factor, σ70 (also called σA in some species), handles housekeeping genes during normal growth. But when conditions change, the cell swaps in alternative sigma factors that redirect RNA polymerase to new promoters.
σ38 (RpoS) takes over during stationary phase or when cells face acid or osmotic stress. σ32 (RpoH) activates heat shock genes when the cell encounters high temperatures, ethanol, heavy metals, or hydrogen peroxide. σ28 (FliA) drives expression of flagella and chemotaxis genes. A large family of ECF (extracytoplasmic function) sigma factors responds to signals like iron availability, antibiotic production, and outer membrane damage.
There’s also an entirely separate sigma family, σ54, which works through a fundamentally different mechanism and requires an activator protein powered by ATP hydrolysis to form the open complex. This two-family system, σ70-type and σ54-type, gives bacteria a flexible toolkit for reprogramming gene expression in response to their environment.
Activators and Repressors at the Promoter
Sigma factors aren’t the only proteins that interact with promoter regions. Regulatory proteins bind nearby to either help or block transcription.
Repressors bind to a region called the operator, which typically overlaps with or sits just downstream of the promoter. The classic example is the lac repressor in E. coli, which binds to an approximately 30-base-pair operator sequence starting a few bases before the transcription start site. When the repressor occupies this space, it physically blocks RNA polymerase from binding or moving forward. This is negative control: the default state is “off” until the repressor is removed (by the presence of lactose, in this case).
Activators work the opposite way. Catabolite activator protein (CAP) binds to a site about 60 base pairs upstream of the transcription start site in the lac operon when it’s bound to cyclic AMP. CAP then makes direct contact with the alpha subunit of RNA polymerase, helping the enzyme bind more tightly to the promoter. This is positive control: the activator’s presence boosts transcription above its baseline level.
Small Molecules That Alter Promoter Binding
Beyond protein factors, small signaling molecules can change how RNA polymerase interacts with promoters. The best-studied example is ppGpp, the alarm signal of the stringent response. When bacteria face amino acid starvation or other nutrient stress, ppGpp levels spike. This molecule binds directly to RNA polymerase at two sites: one at the interface of the ω and β’ subunits, and another at the interface of the β’ subunit and a transcription factor called DksA.
The effect is selective. ppGpp doesn’t shut down all transcription. Instead, it shifts RNA polymerase’s preferences, reducing its affinity for promoters of genes like ribosomal RNA (which the cell no longer needs to produce at high levels during starvation) while increasing transcription of amino acid biosynthesis and stress response genes. The promoter’s specific sequence, DNA curvature, and ease of strand separation all determine whether ppGpp binding to RNA polymerase will enhance or inhibit transcription at that site.