User:Ashleystrenko/sandbox

Bacterial transcription is the process in which a segment of bacterial DNA is copied into a newly synthesized strand of messenger RNA (mRNA) with use of the enzyme RNA polymerase. The process occurs in 3 main steps: initiation, elongation, and termination; and the end result is a strand of mRNA that is complimentary to a single strand of DNA. The transcribed region can account for more than one gene. In fact, many prokaryotic genes occur in operons, which are a series of genes that work together to code for the same protein or gene product and are controlled by a single promoter. Prokaryotic RNA polymerase is made up of four subunits and when a fifth subunit attaches, called the σ-factor, the polymerase can recognize specific binding sequences in the DNA, called promoters. The binding of the σ-factor to the promoter is the first step in initiation. Once the σ-factor releases from the polymerase, elongation proceeds. The polymerase continues down the double stranded DNA, unwinding it and synthesizing the new mRNA strand until it reaches a termination site. There are two termination mechanisms that are discussed in further detail below. Termination is required at specific sites for proper gene expression to occur. Gene expression determines how much gene product, such as protein, is made by the gene.

Prokaryotic transcription differs from eukaryotic transcription in several ways. In prokaryotes, transcription and translation can occur simultaneously in the cytoplasm of the cell, whereas in eukaryotes transcription occurs in the nucleus and translation occurs in the cytoplasm. There is only one type of prokaryotic RNA polymerase whereas eukaryotes have 3 types. Prokaryotes have a σ-factor that detects and binds to promoter sites but eukaryotes do not need a σ-factor. Instead, eukaryotes have transcription factors that allow the recognization and binding of promoter sites.

Initiation
Initiation of transcription requires promoter regions, which are specific nucleotide consensus sequences that tell the σ-factor on RNA polymerase where to bind to the DNA. The promoters are usually located 15 to 19 bases apart and are most commonly found upstream of the genes they control. RNA polymerase is made up of 4 subunits, which include two alphas, a beta, and a beta prime (α, α, β, and β'). A fifth subunit, sigma (called the σ-factor), is only present during initiation and and detaches prior to elongation. Each subunit plays a role in the initiation of transcription, and the σ-factor must be present for initiation to occur. When all σ-factor is present, RNA polymerase is in its active form and is referred to as the holoenzyme. When the σ-factor detaches, it is in core polymerase form. The σ-factor recognizes promoter sequences at -35 and -10 regions and transcription begins at the start site (+1). The sequence of the -10 region is TATAAT and the sequence of the -35 region is TTGACA.


 * The σ-factor binds to the -35 promoter region. At this point, the holoenzyme is referred to as the closed complex because the DNA is still double stranded (connected by hydrogen bonds).
 * Once the σ-factor binds, the remaining subunits of the polymerase attach to the site. The high concentration of adenine-thymine bonds at the -10 region facilitates the unwinding of the DNA. At this point, the holoenzyme is called the open complex. This open complex is also called the transcription bubble. Only one strand of DNA, called the template strand (also called the noncoding strand or nonsense/antisense strand), gets transcribed.
 * Transcription begins and short "abortive" nucleotide sequences approximately 10 base pairs long are produced. These short sequences are nonfunctional pieces of RNA that are produced and then released.
 * The σ-factor is needed to initiate transcription but is not needed to continue transcribing the DNA. The σ-factor dissociates from the core enzyme and elongation proceeds. This signals the end of the initiation phase and the holoenzyme is now in core polymerase form.

Elongation
During elongation, RNA polymerase slides down the double stranded DNA, unwinding it and transcribing (copying) its nucleotide sequence into newly synthesized RNA. New nucleotides that are complementary to the DNA template strand are added to the 3' end of the RNA strand. The newly formed RNA strand is practically identical to the DNA coding strand (sense strand or non-template strand), except it has uracil substituting thymine, and a ribose sugar backbone instead of a deoxyribose sugar backbone. Because nucleoside triphosphates (NTPs) need to attach to the OH- molecule on the 3' end of the RNA, transcription always occurs in the 5' to 3' direction. The four NTPs are adenosine-5'-triphosphate (ATP), guanoside-5'-triphosphate (GTP), uridine-5'-triphosphate (UTP), and cytidine-5'-triphosphate (CTP). The attachment of NTPs onto the 3' end of the RNA transcript provides the energy required for this synthesis. NTPs are also energy producing molecules that provide the fuel that drives chemical reactions in the cell.

Multiple RNA polymerases can be active at once, meaning many strands of mRNA can be produced very quickly. RNA polymerase moves down the DNA rapidly at approximately 40 bases per second. The polymerase has a proofreading mechanism that limits mistakes to about 1 in 10,000 nucleotides transcribed. RNA polymerase has lower fidelity (accuracy) and speed than DNA polymerase. DNA polymerase has a very different proofreading mechanism that includes exonuclease activity, which contributes to the higher fidelity. The consequence of an error during RNA synthesis is usually harmless, where as an error in DNA synthesis could be detrimental.

The promoter sequence determines the frequency of transcription of its corresponding gene.

Termination
In order for proper gene expression to occur, transcription must stop at specific sites. Two termination mechanisms are known:


 * Intrinsic termination (also called Rho-independent termination): Specific DNA nucleotide sequences signal the RNA polymerase to stop. The sequence is commonly a palindromic sequence containing G and C nucleotides. When the polymerase encounters this sequence it causes the RNA transcript to fold back on itself and its G and C nucleotides base pair. This causes a hairpin loop to form and stalls the polymerase. Following the G-C rich region in the RNA termination sequence are a series of U nucleotides that base pair with A nucleotides on the DNA template. The U-A bonds are weak and this, coupled with the stalled polymerase, allows for the RNA transcript to release from the DNA template.
 * Rho-dependent termination: ρ factor (rho factor) is a terminator protein that attaches to the RNA strand and follows behind the polymerase during elongation. Once the polymerase nears the end of the gene it is transcribing, it encounters a series of G nucleotides which causes it to stall. This stalling allows the rho factor to catch up to the RNA polymerase. The rho protein then pulls the RNA transcript from the DNA template and the newly synthesized mRNA is released, ending transcription.

The termination of DNA transcription in bacteria may be stopped by certain mechanisms wherein the RNA polymerase will ignore the terminator sequence until the next one is reached. This phenomenon is known as antitermination and is utilized by certain bacteriophages.