RNA Polymerase

RNA polymerase (RNA-p) is an enzyme that is responsible for the synthesis of RNA in the 5' -> 3' direction using a complementary and antiparallel DNA strand as a template. RNA-p is multimeric enzyme, the core enzyme consists of the four subunits β, β', α, ω. The haloenzyme consists of the core enzyme and the σ factor. It is the sigma factor that is responsible for directing the RNA polymerase to the appropriate start site for RNA synthesis. This means that the σ factor is responsible for recognizing promoters, each specific σ factor interacting with a specific promoter sequence. The σ factor disassociates from the enzyme shortly after the transcription initiation.

Control of Transcription

Transcription is the most common step at which gene expression can be controlled. The proteins responsible for this are DNA interacting proteins, which bind to specific sites on the DNA and can influence the regulation of transcription. These regulatory proteins fall into two catagories:

• Repressors
• Activators

Repressors

Repressors are DNA binding proteins that are responsible for the negative control of transcription. This control is achieved by the binding of the active repressor to an operator site, which results in the RNA-p being unable to instigate RNA transcription.

Activators

Activators are DNA binding proteins that are responsible for the positive control of transcription. The sequence of nucleotide of promoters under positive control typically interact poorly with RNA-p and the activators help the RNA-p recognize the promoter and begin transcription. The activator-binding site can either be found close to the promoter or a distance from it. The activator changes the conformation of the DNA bringing about the additional contacts necessary for RNA-p to initiate transcription.

Transcription Termination

Termination is the process by which the elongation of an RNA molecule is ceased.

Termination can fall into one of two categories:

• Intrinsic Termination
• Factor-dependent Termination

Intrinsic Termination

Intrinsic Termination can be found to occur at defined template sequences, usually a region of hyphenated inverted sequence symmetry followed by a run of T residues. Termination through intrinsic terminators is stimulated by additional factors, e.g. NusA. Termination occurs due to the stem-loop structure formed by the base-pairing of mRNA with itself caused by inverted sequence symmetry, followed by the run of T residues. The NusA protein causes the RNA-p complex to temporarily stall at the stem-loop structure, when this is followed by a poly-A tail, the RNA-DNA duplex is destabilized. This causes the RNA-p to dissociate from the DNA, thereby terminating transcription. Termination functions step-by-step.

Factor dependent Termination

Factor-dependent Termination occurs due to events that are not directly related to transcription, such as the release of ribosomes from nascent transcript or DNA damage. One such host termination factor is Rho, which acts on many sites along the bacterial chromosome. (( ??MFD, is a host termination factor that is responsible for releasing RNA-p stalled at sites of UV-induced DNA lesions. ??)) rho-dependent termination is characterized by not having a specific hairpin structure involved in the termination. The termination thus happens whendue to XXXXXX, and what have been found of the termination site any commen sequences or consensus ????????????????? the function of rho dependent termination, have been shown to be affected by XXXX. The rho binding sites on the mRNA, have been identified from XXbp to XXXbp of stream of the termination site. Rho-termination is thus an example of the more complex termination regulation that is not fully understood and can be very difficult to define and use for engineering purposes. Thus for a more defined anti-termination system the lambda N-protein system and the interaction with the nut-site in the phage genome ????? is a more defined system. Recent research have found out that the Rho termination..?

Phages

Fluorescence Proteins

Fluorescence proteins (FPs) are proteins that are capable of forming visible wavelength chromophores from a sequence of 3 amino acids within their own polypeptide sequence.

Background on measuring

Micro Fermentor System

We used a 48 well microtiter plate fermentor system called BioLector for our characterization experiments.

• potatoes
• spinach
• lollipops

Flow Cytrometry

We used a Flow cytometer for characterization experiments of the trigger point for the antiterminator mechanism of our construct.

Protocol

For a more detailed protocol see the protocol section under notebook.

Antiterminator trigger point
We used the folowing procedure

• Transformation of the SPL intro the reporter plasmid, recovery 1-2h.
• Plate to verify succes, and starting diluted cultures with antibiotics growing for approx 5generations
• measuring on the flow cytometer.

Background litterature and information

For some basic information on Flow cytometry and FACS the folowing papers can be red