Bacterial and Eukaryotic Transcription Introduction

Genetics and Transcription Unit Structure

• Gene Anatomy and the Transcription Unit     - There are specific functional regions in DNA that dictate how and where transcription occurs.     - The Promoter:         - This sequence is located upstream of the site where the actual decoding of DNA into RNA begins.         - It sets the directionality for the protein machinery (RNA polymerase) so that it sits on the correct strand facing the correct direction.         - Important Note: The promoter is NOT transcribed or decoded into the final RNA molecule.         - Impact of Mutations: If the promoter is mutated, proteins cannot recognize the DNA as a gene to be transcribed. A mutant promoter can completely destroy the function of the gene by preventing any transcription from ever occurring.     - The Plus One Site ($+1$):         - This is the first nucleotide/base that is transcribed into the RNA transcript.         - Nucleotides downstream (after the start site) are referred to as plus ($+$, e.g., $+2$, $+3$) based on their position.         - Nucleotides upstream (before the start site) are referred to as minus ($−$, e.g., $-10$, $-35$). These numbers tell you exactly how many nucleotides a site is from the start site.     - RNA Coding Region:         - This is the region of DNA that will literally be converted into the RNA sequence.         - It begins at the $+1$ site and ends at the termination site.         - It often includes portions that may not be translated later, but the entirety of this region is transcribed into the initial RNA transcript.     - Transcription Termination Site:         - This site signals the RNA polymerase to stop its work and release the produced RNA molecule.         - The mechanism is very obvious in bacteria and is a more complicated molecular process in eukaryotes.

The Bacterial RNA Polymerase Complex

• Molecular Machine Construction:     - RNA polymerase is a large protein complex, not a single protein. It is considered a molecular machine made of multiple subunits.     - The Core Polymerase:         - Consists of two alpha ($\text{2}\times\beta$), two beta ($\text{2}\times\beta$), and one omega (\text{1}\times\text{\omega}) subunit.         - This core is responsible for the actual work of polymerization—building the RNA molecule.     - The Sigma Factor (\text{\sigma} subunit):         - This unit is part of the core enzyme but is not necessary for the polymerization (extension) phase.         - It is absolutely necessary for the initiation phase: recognizing the promoter, binding to specific regions, and orienting the polymerase correctly.         - Once the machinery is assembled and transcription begins, the sigma factor is released to be reused by another core enzyme.

Bacterial Promoter Sequences and Recognition

• Key Regulatory Regions:     - In bacteria, the promoter contains two critical consensus regions known as the $-10$ and $-35$ sequences.     - The Minus 10 Region:         - Typically follows the consensus sequence $TAT AAT$.         - This is often referred to as the "Pribnow box," named after the scientist who discovered its importance.     - The Minus 35 Region:         - Another critical consensus sequence that provides a binding site for the sigma factor (\text{\sigma}).     - Binding and Transcription Rates:         - These regions are the most important for starting transcription. Mutations here reduce binding the most, thereby reducing transcription rates.         - The closer a sequence matches the "perfect" consensus, the higher the rate of sigma factor binding.         - Binding is not binary (black and white); it exists on a spectrum from zero binding to optimal binding based on the molecular sequence.         - Concentration Factors: The amount of transcription is also determined by the concentration of transcription factors. For instance, a tenfold increase in sigma factor production can lead to a tenfold increase in the transcription of genes using that factor.     - Convention: Scientists always refer to the non-template strand sequence when discussing consensus sequences and promoters.

Upstream Regulatory Elements

• Beyond the Basal Promoter:     - While the core/basal promoter ($-10$ and $-35$) is essential, other sequences further upstream help regulate how often or how much transcription happens.     - Upstream Elements:         - Found in bacteria usually between $−40$ and $−60$ nucleotides from the start site.         - Enhancer Proteins: Can bind here to change the DNA shape and significantly increase transcription.         - Repressor Proteins: Can bind here to change the DNA shape to inhibit protein access to the gene.

The Process of Transcription: Initiation and Elongation

• Initiation:     - Sigma factor (\text{\sigma}) recognizes the $-10$ and $-35$ sequences.     - The holoenzyme (Core + Sigma) assembles at the proper location.     - RNA polymerase parts denature the DNA double helix to expose the template.     - De Novo Synthesis: Unlike DNA polymerase, RNA polymerase can start a new strand from scratch without a primer.

• Chemical Mechanism:     - The first base of the RNA transcript retains its three phosphate bonds (triphosphate form).     - Subsequent nucleotides come in as triphosphates; RNA polymerase catalyzes the phosphodiester bond and releases two phosphates (pyrophosphate).

• Elongation:     - After a handful of bases are produced, the sigma factor (\text{\sigma}) is released.     - The Core Enzyme continues on its own.     - Speed: Transcription occurs at approximately $40\,\text{nucleotides per second}$.     - Transcription Bubble: A stretch of $10$ to $20$ nucleotides exists as a DNA-RNA hybrid within the moving polymerase.     - Polymerase Responsibilities:         - Topoisomerase activity: Unwinding the helix ahead and rewinding it behind.         - Helicase activity: Denaturing the double-stranded DNA.         - Polymerase activity: Building the RNA strand.

• Regulation During Elongation:     - DNA modifications can impede the movement of the polymerase. Transcription may pause until these modifications are removed.

Transcription Termination in Bacteria

• Mechanisms of Termination:     - There are two primary mechanisms in bacteria: Rho-independent (intrinsic) and Rho-dependent.

• Intrinsic (Rho-Independent) Termination:     - This is coded within the DNA sequence of the gene itself and is part of the RNA molecule.     - Inverted Repeats: The sequence contains inverted repeats that, when transcribed into RNA, form a complementary stem-loop or hairpin structure.     - Poly-U Tail: Immediately following the stem-loop sequence is a stretch of Adenines ($A$) in the template DNA, which results in a stretch of Uracils ($U$) in the RNA.     - Biochemical Strain: The stem-loop is a rigid physical structure. The hybrid between state and the Poly-U stretch consists only of $2$ hydrogen bonds per base (AU pairs), which are weaker than GC pairs ($3$ hydrogen bonds).     - The physical strain of the hairpin next to the weak AU hybrid causes the RNA to separate and destabilize, forcing RNA polymerase to release the DNA.

• Rho-Dependent Termination:     - Requires the Rho protein (\text{\rho}), which functions as a helicase.     - Rut Site: The RNA molecule contains a "Rho Utilization" ($rut$) sequence.     - The Rho protein binds to the $rut$ site and travels along the RNA toward the polymerase.     - When it reaches the RNA-DNA hybrid, it uses its helicase activity to denature the hybrid, forcing the polymerase to let go.

• Non-Reversibility:     - Once termination occurs and the polymerase is gone, it cannot just restart at that spot; it must be brought back to a promoter by a sigma factor (\text{\sigma}).

Questions & Discussion

• Q: Is the promoter sequence transcribed?     - A: It is not. The promoter is absolutely part of the gene, but it is not part of the RNA transcript. The RNA coding region typically begins right after the promoter.

• Q: How do we describe mutation locations?     - A: We use the plus/minus numbering system. For example, a "C to G at 123" mutation tells you the exact position relative to the transcription start site.

• Q: How does template strand orientation affect the RNA sequence?     - A: DNA is read $3' \rightarrow 5'$ and RNA is built $5' \rightarrow 3'$. Because they must be anti-parallel, if the DNA template is provided in a $5' \rightarrow 3'$ format, the RNA sequence must be determined by looking at the complements in the opposite direction.     - Example: If DNA template is $5'-G-T-C...$, then at the $5'$ end of the DNA (which is the $3'$ end of the relative alignment), the RNA will have the complement $C$ at its $3'$ end.

• Q: Comparison between domains?     - A: Archaea undergo these processes more similarly to Eukaryotes than to Bacteria. Eukaryotic transcription will be covered in the next session.