ÓRGANOS CON FUNCIONES DE CONTROL

cell identity and function by prokaryote-style repressors would be very wasteful. However, there are some DNA sequences that are transcribed without the need for transcription factors as they contain a promoter site which directly induces RNA polymerase binding and transcription.91 _{These sequences typically encode for proteins}

that need to be constantly expressed for cell function, but they are often present at different concentrations at different stages of the cells lifetime with their expression level tightly controlled by repressors.

Prokayrotes contain a single DNA-dependent RNA polymerase, a 390 kDa complex consisting of 5 core subunits.92 _{Together with the transcription factor, these six sub-}

units make up the RNA polymerase holoenzyme. Unlike its eukaryotic equivalents, which pause when a mismatched base is added and can then reverse and remove errors, this complex does not contain a separate proof-reading subunit. The polymerase is therefore relatively error-prone, inserting the wrong base pair roughly every 105 _{to 10}6

bases. This limited fidelity is less critical for RNA than for DNA synthesis as mRNA is eventually broken down and the nucleotides recycled. The genome in prokaryotic cells is much smaller than that of the eukaryotes and as they lack a nucleus, transcription of DNA and translation of mRNA both occur in the cytoplasm. Genes encoding metabolic pathways in prokaryotes are typically found grouped together in the DNA. This allows expression of the entire group in a single transcript and leads to a single point of control over a range of proteins.

Eukaryotes contain three RNA polymerases; RNA polymerase I binds directly to promoters in the gene sequence to synthesise the RNA component of ribosomes and polymerase III makes tRNA and some small specialized RNAs. RNA polymerase II is the general purpose polymerase; it is much more complex than the E. coli polymerase but they share some highly conserved structural features. This polymerase is not active in the absence of a transcription factor.

2

2..11..11 PPrrookkaarryyoottiicc ttrraannssccrriippttiioonn ccoonnttrrooll

The extent of protein expression is controlled by the availability and activity of mRNA with transcription factors controlling the rate at which the RNA polymerase is recruited to particular promoter sequences to produce mRNA. Transcription is principally controlled by moderating the binding of the transcription factor to the promoter region

Zinc finger peptides

of the DNA or to the RNA polymerase. For example, the sigma 70 subunit (a prokaryotic transcription factor) of E. coli RNA polymerase binds a conserved DNA sequence at several positions before the start site of transcription. The spacing of these binding positions define the E. coli transcription start position; the first is at -10 with respect to the start of transcription at a 5`-TATAAT-3` sequence and the second is found at position -35 at a 5`-TTGACA-3` sequence. These sites are highly conserved but do show some variation between different strains of E. coli. Variation from the consensus sequence among E. coli genes can also impose transcription control, as a single site mutation can affect the binding of the sigma 70 subunit and vary gene expression by several orders of magnitude.92 _{Other proteins can either activate or repress}

transcription by binding to these consensus sequences. For example, when glucose levels are low cAMP is formed that binds to the catabolite activator protein. This protein can bind to promoters within the lac operon to increase the transcription of enzymes necessary for the digestion of lactose, but this site is blocked by the lac repressor protein which binds to the lac promoter site between the RNA polymerase binding sites preventing RNA polymerase binding. If lactose is present, it binds to the lac repressor protein which then dissociates from the DNA, allowing transcription to occur. This combination of interlocked regulatory proteins ensures that enzymes necessary to metabolise lactose are only produced when glucose is absent and lactose is present. Transcription can also be repressed by proteins binding further downstream blocking the RNA polymerase as it precesses along the DNA

2

2..11..22 EEuukkaarryyoottiicc ttrraannssccrriippttiioonn ccoonnttrrooll

In eukaryotic cells expression is mainly controlled by the affinity of transcription factors for their DNA target and their affinity for, and ability to, organise complexes with the RNA polymerase holoenzyme. Transcription is also strongly influenced by the acetylation or deacetylation of the histone proteins that DNA is wound around. Acetylation reduces the affinity of DNA to histone proteins increasing the availability of sequences at these sites to RNA polymerases. Conversely, deacetylation increases binding of DNA to histone proteins reducing the availability of sequences to RNA polymerases. Transcription factors can also recruit cofactor or corepressor proteins to the DNA to increase or decrease mRNA production.

Zinc finger peptides

43

Figure 51. DNA -helix.

In order for transcription to occur, the RNA polymerase holoenzyme needs to bind to the DNA and form an active complex. DNA has three major helical forms, the most common of which is the “B DNA helix” which features 2 grooves (Figure 51). The minor grove exposes the glyosidic bonds of the sugar phosphate backbone and the major groove exposes the edges of the bases.92 _{In B DNA the minor grove is 5 Å wide and 8 Å}

deep, not large enough to accommodate an -helix or other protein secondary structure elements, and mainly exposes functionality conserved by all DNA bases. In contrast, the major grove is 12 Å wide and 8 Å deep, large enough cavity to fit an - helix, and contains base specific hydrogen bond acceptor and donor patterns that allow bases to be recognised without the need for significant a conformational change of the DNA helix.

Zinc finger peptides

2.2 Zinc finger transcription factors

The class of transcription factors examined during this project are called zinc finger transcription factors. Zinc finger transcription factors (ZF-TF) are a wide-ranging class of proteins that variously regulate individual genes or clusters of related genes. For example, the Snail family of ZF-TFs are thought to play a critical role in cell myogenesis.93 _{These transcription factors contain a number of individual zinc fingers}

(Figure 52), usually between four and six.

Figure 52. A single Cys2His2 zinc finger is shown in blue with the sidechains of two cysteine and two

histidine residues coordinating a zinc ion (green).

Shown in Figure 52 is a single Cis2His2 type zinc finger made up of two short peptide

strands and an -helix in a fold known as a --helix held together by the coordination of the zinc ion from which the fingers derive their name. The zinc ion is coordinated to the sidechains of two histidine residues of the -helix and two cysteine residues, one from each of the short peptide strands. In the absence of a zinc ion the peptide does not form an -helix and shows little if any binding affinity for DNA. Each individual zinc finger unit coordinates to three bases in an E-box transcription factor binding site. These three base pairs in the DNA are not sufficiently selective on their own but when between four and six zinc fingers are bound together in a chain, enough bases are contacted to select unique sites within the genome. Although there is some overlap between the DNA bases contacted by adjacent fingers, this can be seen as a modular design and has allowed the construction of functioning artificial zinc finger proteins

Zinc finger peptides

45

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Zinc fingers have other uses besides transcription factors. When coupled to an otherwise non-selective restriction endonuclease such as the catalytic domain of FokI the resulting construct was used to reduce the expression of mutant Huntingtin protein, one of the major factors in Huntington’s disease, in mice.95

Figure 53. Zinc finger based FokI dimer binding.96

FokI restriction nuclease monomers are inactive and weakly associating and must form a dimer to cut DNA strands. The zinc finger binding to the DNA allows FokI to form an active homodimer at sites defined by the selectivity of the fused zinc finger and create double strand breaks (DSB, Figure 53). These fusions of zinc finger domain and a non-selective endonuclease are called zinc finger nucleases (ZFN).21,97 _{These breaks}

can then be repaired by the error-prone non-homologous end joining (NHEJ).98 _Repair

of DSB caused by ZFN can be used to effectively introduce mutations at the site of the double strand breaks.99 _{This error-prone repair can result in frame shifts in the coding}

DNA causing destruction of a start codon, stop codon or simply generating an incorrect protein product. The outcome of repaired DSB can be influenced by exogenously introduced DNA matching the overhang at the cleavage site allowing the introduction of designed DNA oligonucleotides into genes.99 _{Using tailored zinc fingers it is}

theoretically possible to selectively cleave at unique sites on the genome, but practically this technique is limited to palindromic target sequences by off target effects. However, it is possible to use two individual monomers with oligate heterodimer FokI catalytic domains, eliminating the need for a palindromic sequence and diminishing off-target effects, although it requires the identification of at least eight synergistic zinc

Zinc finger peptides

fingers.96,98 _{Zinc fingers like these have been shown to be intrinsically cell penetrating,}

avoiding a common pitfall of peptide therapeutics.98