DNA Technology Lect 6

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    Base alterations and base damage

    The bases of DNA are subject to spontaneous structural alterations

    called tautomerization.

    If during DNA replication, G is in the enol form, the polymerase will add aT across from it instead of the normal C because the base pairing rules

    are changed (not a polymerase error).

    The result is a G :C to A :T transition; tautomerization causes transition

    mutations only.

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    Another mutatgenic process occurring in cells is spontaneous base

    degradation. Thedeamination of cytosine to uracil happens at a

    significant rate in cells.

    Deamination can be repaired by a specific repair process which detects

    uracil, not normally present in DNA; otherwise the U will cause A to be

    inserted opposite it and cause a C:G to T:A transition when the DNA is

    replicated.

    Deamination of cy tos ine creates uraci l

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    Deamination of methylcytosine to thymine can also occur.

    Methylcytosine occurs in the human genome at a sequence

    which is normally avoided in the coding regions of genes.

    If the meC is deaminated to T, there is no repair system which

    can recognize and remove it (because T is a normal base in DNA).

    This means that wherever the sequence containing meC occurs

    in genes it is a "hot spot" for mutation.

    Deamination of 5-methyl cytosine creates thym ine

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    DNA is damaged by alkylation, oxidation, and radiation

    In alkylation, methyl or ethyl groups are transferred to reactive sites

    On the bases and to phosphates in the DNA backbone.

    Alkylating chemicals include nitrosamines and N-methyl-N-nitro-N-

    Nitrosoguanidine.

    Example: is the formation of O6

    -methylguanine, often mispairs with thymine,resulting in the change of a G:C to A:T when damaged DNA is replicated

    Specif ic si tes on guanine

    that can be damaged b y

    alkylat ion, oxidat ion o r

    radiat ion

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    Ultrav io let induces the format ion o f a cyclobu tane

    r ing between adjacent thym ines

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    The copying of genetic information from DNA ;into messenger RNA is

    the initial step in the chain of reactions leading to synthesis of the

    multitude of proteins and specialized RNA molecules needed by cells.

    The absence of a nuclear membrane is a characteristic of bacteria that

    has a profound effect on transcription.

    Bacterial transcripts are processed rapidly, and their 5' ends oftenenter ribosomes and are directing protein synthesis, while the 3' ends of

    the genes are still being transcribed.

    In contrast, most eukaryotic RNA transcripts must be processed and

    transported out of the nucleus before they can function.

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    Cells make four principal kinds of RNA: ribosomaI (r RNA), transfer(tRNA), messenger (mRNA), and a variety of small RNAs.

    The last, which range in from a few up to several hundred nucleotides

    are designated variously as sRNAs, ncRNAs, miRNAs, siRNAs,

    snRNAs, and snoRNAs.

    The abbreviations s, nc, mi, si, sn, and sno stand for small, non-

    coding, micro, silencing, small nuclear, and small nucleolar,

    respectively.

    All of these RNAs are synthesized as larger transcripts, which

    undergo cleavage and other modifications within the cell.

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    The fact that purified RNA polymerases can synthesize RNA from the

    four ribonucleoside triphosphates using ssDNA as the template

    suggested that transcription, like DNA replication, involves base pairing.

    When dsDNA served as the template, free ssRNA was formed.

    Thus, it appeared likely that at the site of the polymerase action, the

    dsDNA was momentarily pulled apart into single strands and that one of

    these was copied by the polymerase.

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    1. The lac Operon

    Based on studies of the induction of enzymes in bacteria scientists

    proposed the operon model, an operon is a regulated cluster of genes.

    Three structural genes encode the amino acid sequences of b-

    galactosidase (lacZ), permease (lacY), and a transacetylase

    (acetyltransferase, lacA), which transfers acetyl groups from acetyl-CoA

    to b-galactosides.

    These three genes function as a transcriptional unitofthe operon,

    which encodes a single molecule of mRNA.

    Each operon is controlled by a segment of the DNA molecule located at

    the beginning of the operon, i.e., at the 5' end of the coding chain or 3' end

    of the template chain. The first part of this control regionis called the

    promoter (P).

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    The promoter is the site of the initial binding of the RNA polymerase to

    the DNA.

    The rates of association and of initiation of transcription may beinfluenced strongly by various other proteins.

    One of these, the catabolite gene activator protein (CAP; also

    called cAMP receptor protein, CRP), is important to the lac operon. It

    also binds in the promoter region and stimulates transcription.

    Immediately adjacent to the promoter is the operator (O),which is a

    binding site for a represser (R).

    When the operator is free, transcription is initiated and proceeds

    through the operator region and on to the genes coding for the threeproteins. On the other hand, if the represser is bound to the operator,

    transcription is blocked.

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    The lac operon is ordinarily subject to repression and is activated by

    the presence of aninducer, now known to be allolactose.

    In the presence of the inducer a conformational change takes place,

    destroying the affinity of the represser protein for the operator site.

    Thus, in the presence of inducer the operator is not blocked, and thetranscription takes place. Such an operon is said to be negatively

    controlled andinducible.

    Important to the control of the operon is the regulatory gene, which

    codes for the synthesis of the repressor protein. In the case of the lac

    operon, the regulatory gene (the I gene) is located immediately

    preceding the operon itself.

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    An operon is a regulated clu ster of genes. This is the lac operon o f E. col i .

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    2- Initiation of transcription

    The rate of RNA synthesis varies from one operon to another.

    Sequences of promoters, operators, and other control sequences aswell as the state of repressor and activator proteins all affect these

    rates.

    In every instance the first steps in transcription involve the binding of

    RNA polymerase to DNA.

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    Bacteria have only one single RNA polymerase (RNAP), ex., E. coli

    RNAP consists of four subunits with the composition bb(core enzyme)and a sigma subunit (s).The three dimensional structure of the E. coliRNAP bound to DNA in

    an initiation complex shows that the enzyme forms a groove into

    which the DNA can fit.

    Of the RNA polymerase subunits s(sigma) plays a unique role in

    initiation of transcription.

    It is required for the recognition of promoter sites.

    It is not needed for elongation of an RNA chain and dissociates from the

    a2bb core complex soon after transcription is initiated.

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    Hypothetical structure of a transcription bubble formed by RNAP.

    This conta ins a shor t DNA-RNA hybr id h elix formed by the growing RNA.

    The DNA double helix is undergoing separation at point A as is the

    hybrid helix at point B.

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    Promoters

    -10 region - consensus of TATAATin E. coli(Pribnow sequence, orPribnow box).

    Polymerization begins 8-10 nucleotides. downstream of -10 region

    -10 region necessary for initial denaturation of DNA to expose template

    and region of contact for core RNA polymerase (no sigma), A-T rich.

    -35 region - consensus of TTGACAin E. coli: necessary for recognition by

    sigma factor.

    An additional DNA element that binds RNAP is found in some strongpromoters, ex. those directing the expression of rRNA genes. This is called

    UP-element.

    http://www.science.siu.edu/microbiology/micr460/460%20Pages/promoter.htmlhttp://www.science.siu.edu/microbiology/micr460/460%20Pages/promoter.html
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    Bacter ial prom oters

    TATAATTTGACA

    Start

    TAC

    3

    3

    5

    5

    TATAATTTGACA

    StartTAC

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    The initiation reaction

    A promoter not only locates the site of initiation but also determines

    the direction of transcription, therefore, the strand of the DNA duplexthat is to serve as a template.

    The requirement for two specific recognition sequences ensures this

    directionality (-10 and -35 sites)

    The RNAP binds to promoter sites through specific interactions with the

    major groove of the DNA.

    The initial specific polymerase-promoter complex is referred to as

    a closed complex.

    In a rate-limiting step, the closed complex is converted into open complex

    which is ready to initiate mRNA synthesis.

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    In the open complex, the hydrogen bonds holding together the base

    pairs have been broken, and the bases of the template chain are available

    for pairing with incoming ribonucleoside triphosphates.

    The RNAP and the dsDNA bind reversibly to form a complex (closed complex)

    with formation constant Kf.

    Pribnow sequence is A-T rich, therefore, opening of the helix at this point

    is easier than in a GC-rich region.

    Thus, Pribnow sequence may represent a point of entry of RNAP to form

    the open complex.

    Kf KE (Polymerase) + P (Promoter) EP (Closed)

    EP (Open) Transcription.

    Kf = rate of formation of closed complex.

    K = rate of formation of open complex.