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 a T 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.

 Another mutatgenic process occurring in cells is spontaneous base degradation. The deamination 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 cytosine creates uracil

 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 thymine

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-NNitrosoguanidine.

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

Specific sites on guanine that can be damaged by alkylation, oxidation or radiation

Ultraviolet induces the formation of a cyclobutane ring between adjacent thymines

 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 often enter 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.

 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, noncoding, 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.

 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.

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 bgalactosidase (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 unit of the 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 region is called the promoter (P).

 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 be influenced 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 three proteins. On the other hand, if the represser is bound to the operator, transcription is blocked.

 The lac operon is ordinarily subject to repression and is activated by the presence of an inducer, 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 the transcription takes place. Such an operon is said to be negatively controlled and inducible. 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.

An operon is a regulated cluster of genes. This is the lac operon of E. coli.

2- Initiation of transcription

The rate of RNA synthesis varies from one operon to another. Sequences of promoters, operators, and other control sequences as well 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.

 Bacteria have only one single RNA polymerase (RNAP), ex., E. coli RNAP consists of four subunits with the composition a2bb (core enzyme) and a sigma subunit (s).

The three dimensional structure of the E. coli RNAP 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.

Hypothetical structure of a transcription bubble formed by RNAP. This contains a short DNA-RNA hybrid helix formed by the growing RNA. The DNA double helix is undergoing separation at point A as is the hybrid helix at point B.

Promoters
-10 region - consensus of TATAAT in E. coli (Pribnow sequence, or Pribnow 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 TTGACA in E. coli: necessary for recognition by sigma factor. An additional DNA element that binds RNAP is found in some strong promoters, ex. those directing the expression of rRNA genes. This is called UP-element.

TTGACA

TATAAT

3
Start TAC

TTGACA

TATAAT

3
Start TAC

Bacterial promoters

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 duplex that 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.

 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. E (Polymerase) + P (Promoter) EP (Open) Kf EP (Closed) Transcription. K

Kf = rate of formation of closed complex. K = rate of formation of open complex.