15.2 Prokaryotic Transcription

Prokaryotic Transcription

Prokaryotic transcription is the process by which bacterial cells copy DNA into RNA. This RNA then serves as the instructions for building proteins. The process has three main stages: initiation, elongation, and termination. Because prokaryotes lack a nucleus, transcription happens directly in the cytoplasm, and translation can actually begin on the mRNA while it's still being transcribed.

Promoters and termination signals are the key regulatory elements here. Promoters control when and how frequently a gene gets transcribed, while termination mechanisms make sure the RNA polymerase stops at the right place. Together, these allow bacteria to respond rapidly to environmental changes.

Steps of Prokaryotic Transcription

1. Initiation

This is where transcription begins. RNA polymerase must find and bind to the correct spot on the DNA before any RNA can be made.

• The sigma factor ($\sigma$), a subunit of RNA polymerase, recognizes and binds to the promoter region located upstream of the gene. The promoter contains two conserved consensus sequences: the -10 box (TATAAT, also called the Pribnow box) and the -35 box (TTGACA). These numbers refer to their position relative to the transcription start site (+1).
• Once RNA polymerase binds the promoter, the DNA double helix unwinds locally, forming a transcription bubble of about 12-14 base pairs. This exposes the template strand so RNA synthesis can begin.

2. Elongation

Once initiation is complete, the sigma factor is released, and the core enzyme continues on its own.

• RNA polymerase reads the template strand in the 3' to 5' direction and builds the new RNA strand in the 5' to 3' direction.
• Ribonucleotides (ATP, UTP, CTP, GTP) are added according to base-pairing rules: A pairs with U, T pairs with A, G pairs with C, and C pairs with G.
• RNA polymerase catalyzes phosphodiester bonds between each incoming nucleotide and the growing RNA chain.
• The enzyme continues synthesizing RNA until it encounters a termination signal on the DNA.

3. Termination

Transcription must stop at the right place. Prokaryotes use two distinct mechanisms to accomplish this: Rho-dependent and Rho-independent termination (covered in detail below).

Promoters in Prokaryotic Gene Regulation

Promoters are DNA sequences upstream of the transcription start site that determine whether and how often a gene gets transcribed. RNA polymerase must bind the promoter to begin transcription, so the promoter acts as a gatekeeper for gene expression.

Promoter strength depends on how closely the -10 and -35 sequences match the consensus:

• Strong promoters closely match the ideal TATAAT and TTGACA sequences. These drive high rates of transcription because RNA polymerase binds them efficiently.
• Weak promoters deviate from the consensus, so RNA polymerase binds less frequently, resulting in lower transcription rates.

Regulatory proteins fine-tune transcription by interacting with or near the promoter:

• Activator proteins enhance transcription by helping recruit RNA polymerase to the promoter. For example, the CAP protein (catabolite activator protein) binds near the lac promoter when glucose is low, increasing transcription of genes needed to metabolize alternative sugars like lactose.
• Repressor proteins block transcription by preventing RNA polymerase from binding or moving forward. The lac repressor, for instance, binds the operator region next to the lac promoter and physically blocks RNA polymerase when lactose is absent.

Environmental signals like nutrient availability or temperature shifts influence whether these regulatory proteins are active or inactive. This is how bacteria adapt their gene expression to changing conditions.

Prokaryotic Transcription Termination Mechanisms

Rho-Dependent Termination

This mechanism requires a helper protein called Rho ($\rho$), which is an ATP-dependent RNA helicase.

1. Rho binds to a specific sequence on the nascent RNA called a rut site (Rho utilization site).
2. Using energy from ATP hydrolysis, Rho translocates along the RNA in the 5' to 3' direction, chasing the RNA polymerase.
3. When RNA polymerase pauses (often at a GC-rich region), Rho catches up and uses its helicase activity to unwind the RNA-DNA hybrid within the transcription bubble.
4. This causes RNA polymerase to dissociate from the DNA, releasing the newly made RNA.

Rho-Independent (Intrinsic) Termination

This mechanism doesn't need any extra proteins. The termination signal is built right into the DNA sequence.

1. As RNA polymerase transcribes the terminator region, the nascent RNA forms a GC-rich hairpin loop (a stem-loop secondary structure).
2. Immediately downstream of the hairpin, a stretch of uracil residues (from a poly-A sequence in the template) creates weak A-U base pairs between the RNA and DNA.
3. The combination of the hairpin causing RNA polymerase to stall and the weak A-U pairing destabilizes the transcription bubble.
4. RNA polymerase dissociates from the DNA, and the RNA transcript is released.

RNA Polymerase and DNA Strands in Transcription

Prokaryotic RNA polymerase exists in two forms:

• Core enzyme: Contains the catalytic subunits responsible for RNA synthesis (subunit composition: $\alpha_2\beta\beta'$).
• Holoenzyme: The core enzyme plus the sigma factor. The sigma factor is only needed for promoter recognition during initiation. Once elongation begins, sigma dissociates, and the core enzyme carries out the rest of transcription.

During transcription, RNA polymerase interacts with two DNA strands:

• Template strand (antisense strand): The strand that RNA polymerase actually reads. The RNA is built as a complement to this strand.
• Coding strand (sense strand, non-template strand): This strand has the same base sequence as the resulting RNA, except with thymine (T) instead of uracil (U). You won't see RNA polymerase directly reading this strand, but it's useful for predicting the mRNA sequence.

Ribonucleotides (the building blocks of RNA) are added one at a time to the 3' end of the growing chain during elongation, each joined by a phosphodiester bond.