The central dogma of molecular biology postulates the formation of polypeptides and proteins from DNA by two processes called transcription and translation. The biological meaning of these processes, united by the name of protein biosynthesis, is the creation of protein structures in the body. These proteins are used from different angles: they can be enzymes, building and structural proteins, receptors and signaling proteins, as well as transport, storage, and regulatory molecules — the variety of uses of proteins within the body is enormous. It is clear that the organism must be able to produce proteins on its own because not always the materials coming with food can be effectively absorbed by the organism. In addition, it is not always possible for an individual to obtain proteins with food since there are problems of starvation or an unbalanced diet. Consequently, the eukaryotic organism must be able to create its own proteins that perform autonomous tasks. Protein biosynthesis is the solution to this problem, and one of the two fundamental steps in this process is transcription.
In a general sense, transcription should be understood as the process of translating DNA into mRNA. The nucleotide sequence of DNA, roughly speaking, is a collection of four nucleotides, namely adenine (A), guanine (G), cytosine (C), and thymine (T), complementarily linked through hydrogen bonds. The unique sequences of these nitrogenous bases form the genetic code, in which all information about protein-coding is encoded. In fact, it is essential to recognize that the genetic code contains information not only about the structure of future proteins but also includes the regulatory parts of an expression, including introns that are missing from mRNA after transcription. For a number of reasons related to the size of DNA molecules and the evolutionary mechanism of protein formation, DNA cannot take part in protein synthesis directly, so the main genetic molecule requires an intermediary, the role of which is played by mRNA. Thus, as a result of transcription on the antisense DNA matrix, mRNA is formed, which is a complementary copy of the parental nucleotide sequence.
The transcription process is much more complex than described in the previous paragraph. In addition to the reactant (DNA) and product (mRNA), intranuclear transcription involves the use of enzymatic proteins, namely DNA-dependent RNA polymerases. In addition, transcription requires the presence of a promoter, a relatively short strand of DNA located upstream of the initiation site of the process. It is with the promoter that RNA polymerase binds, and the site location is determined by the presence of a TATA box to which the transcription factors that initiate the entire process of mRNA synthesis attach. It is fair to say that not every eukaryote promoter necessarily contains a TATA box.
Transcription factors should be understood as any proteins that are capable of regulating the processes of transcription by attaching to DNA chains. It is clear that such factors can both catalyze this reaction and inhibit it, depending on the organism’s need for a particular type of protein. Consequently, by attaching to specific recognition sites and uniquely interacting with DNA or RNA polymerase, transcription factors are able to control whole gene expression within the cell. Nevertheless, it makes little sense to use a specific transcription factor explicitly since any loss or breakage of its synthesis mechanisms would lead to further inability to regulate gene expression; therefore, transcription factors often act in combination, exerting a cumulative effect on the regulation of transcription.
In general, the need to regulate gene expression is driven by the different protein requirements of cells. Since somatic cells throughout the body have identical genes, it makes no sense to produce molecules of, say, trypsin in skin cells or brain cells since they will not perform the correct functions there. For this reason, the regulation of expression by transcription factors is necessary to ensure the normal functioning of body systems; through the combined effect of such factors, the regulation of transcription is optimized.
One variant of the combined effect of transcription factors is their co-attachment to enhancers that activate mRNA synthesis processes. For example, the enhancer that regulates immunoglobulin heavy chain synthesis consists of more than two hundred nucleotides, so up to nine transcription factors can attach to it (Cooper, 2020). Notably, the combined activity of these factors contributes to rapid immunoglobulin production, but the removal of any of them does not prove critical for the organism since their functions are often duplicated.
Another illustrative example is the expression of estrogen ERα receptors in eukaryotic cells. ERα is a significant type of estrogen hormone receptor, the presence of which positively affects the functioning of puberty and pregnancy in individuals. Among other things, ERα itself is a ligand-dependent transcription factor that can efficiently bind to other factors. Campbell et al. (2018) report two transcription factors, NFIB and YBX1, that are able to bind to the ERα receptor to inhibit breast cancer risk in women simultaneously. More specifically, ERα is a therapeutic target for this type of cancer, and depending on what the estrogen receptor interacts with, there can be a positive or negative tendency for breast cancer to develop in the body. In other words, in this case, it is appropriate to say that the same transcription factor is capable of leading to different regulatory results depending on what it has been associated with.
Another example of the combined action of transcription factors is the fine-tuning of factors in order to use the regulation of expression for therapeutic purposes. First and foremost, transcription factors consist of a DNA-binding domain, which recognizes the DNA strand, and a transcription activation domain. The combined effect of the two transcription factors can be that one of them replaces the DNA-binding domain of the other, as was shown for LexA, which replaced the corresponding domain in Gal4 (Becskei, 2020). The result is the formation of a hybrid transcription factor that rapidly activated transcriptional expression in yeast. In other words, the combined interaction of transcription factors, in this case, is due to the enhancement or suppression of the regulatory activity of such hybrid complexes, which allows not only switching on but also enhancement of DNA-based mRNA expression as a result of transcription.
DNA replication is the most important process in the regulation of cell life, contributing to efficient cell division and preservation of genetic material through generations. The basic pattern of replication consists of the initial breaking of hydrogen bonds between the nucleotides that form the DNA and the subsequent construction of new strands. Thus, from two chains of parental DNA, two more daughter DNA chains are formed as a result of replication: the number of DNA molecules in the organism doubles. This is critical for mitotic or meiotic division when the DNA must be equally divided between the resulting cells so that the new individuals retain at least a haploid set of species genes. However, the complexity of replication is a serious drawback to the smoothness of this process because contingencies can arise; among these, discontinuities are essential to discuss.
The first rupture variant is the nick in DNA, which is formed as a result of a break in the phosphodiester bond between neighboring nitrogenous bases. It is assumed that the leading cause of such a nick is enzymatic, nicking enzyme, activity during replication, and it is noteworthy that gap formation is not necessarily undesirable. For example, topoisomerase I deliberately cuts one of the strands in DNA in order to induce relaxation of strongly twisted helices, which facilitates more reliable reading of the nucleotide sequence by DNA polymerase. It is noteworthy that nick formation in DNA is used for laboratory purposes because labeled Okazaki fragments are formed with this help. These tags retain their integrity for further recognition by DNA ligase.
Another form of breakage during replication is a block during fork movement, preventing the formation of the correct copies. Usually, the two daughter strands of DNA are completed in the opposite direction to each other, and when they meet the replication fork or the end of the chromosome, replication stops. However, in the case of unstable operation of such a fork, a fork reversal occurs, as a result of which the daughter DNA starts to be docked on its complementary second daughter DNA, which disrupts the semantic integrity of replication. On the other hand, fork reversion can be considered as a protective process against damaged replication forks, which allows the genome to remain stable during genotoxic stress. In this case, the formation of such reversible forks is conditioned by the activity of helicase enzymes or the translocase activity of SNF2 protein complexes (Kolinjivadi et al., 2017). It has been reported that reversible forks can be repaired after DNA damage is repaired. In other words, such forks should be considered as temporary states that allow the replisome to bypass damaged restriction sites.
However, it is clear that DNA breaks during replication are not a desirable phenomenon outside the replication processes because they imply disruption of the physical integrity of the genome and the appearance of fragmented sequences. For this reason, the cell needs nick repair and reverse fork repair mechanisms that reduce mutagenesis during DNA polymerase skipping during replication. The primary recombinant practice of such repair is a multistep procedure of sequential gap repair. To do this, the broken parental chain is initially lengthened, after which the replication fork is reversed, as it is initially done in the chicken foot form of reversion. As a result of this process, the leading daughter DNA builds up on the newly synthesized lagging daughter chain, the subsequent removal of which helps restore the replication fork. A similar recombinant repair can be accomplished by the eukaryotic proteins Rad51/Rad52, which anneals complementary DNA chains (Sabatinos, 2020). In contrast to eukaryotic cells, prokaryotic cells have developed several fundamental mechanisms for restarting replication forks that have encountered barriers in order to restore genomic integrity. One such mechanism is the use of recombinant PriA, PriB, or PriC proteins that can detect and remodulate DNA breaks in Escherichia coli (Windgassen et 2018). The mechanism of such repair is based on a replicative DNA helicase reset, which allows cutting the daughter DNA already created and synthesizing a new, already correct strand.
References
Becskei, A. (2020). Tuning up transcription factors for therapy. Molecules, 25(8), 1-19.
Campbell, T. M., Castro, M. A., de Oliveira, K. G., Ponder, B. A., & Meyer, K. B. (2018). ERα
binding by transcription factors NFIB and YBX1 enables FGFR2 signaling to modulate estrogen responsiveness in breast cancer. Cancer Research, 78(2), 410-421.
Cooper, G. M. (2020). Regulation of transcription in eukaryotes. NIH. Web.
Kolinjivadi, A. M., Sannino, V., De Antoni, A., Zadorozhny, K., Kilkenny, M., Técher, H.,… & Costanzo, V. (2017).
Smarcal1-mediated fork reversal triggers Mre11-dependent degradation of nascent DNA in the absence of Brca2 and stable Rad51 nucleofilaments. Molecular Cell, 67(5), 867-881.
Sabatinos, S. A. (2020). Recovering a stalled replication fork. Nature. Web.
Windgassen, T. A., Wessel, S. R., Bhattacharyya, B., & Keck, J. L. (2018). Mechanisms of bacterial DNA replication restart. Nucleic Acids Research, 46(2), 504-519.