Biochemistry, Replication and Transcription

The flow of genetic information in biological systems from DNA>RNA>Protein is the central dogma in molecular biology. This explains how the genetic information in the form of DNA in a cell is converted to RNA and then to protein for effective utilization.

The process of replication allows cells to generate new genetic material (DNA) using original DNA as a template. The cell cycle consists of four phases-G1, S, G2, and M. During the G1 phase, cells grow and produce material like nucleotide precursors as preparation for DNA replication in the S-phase. Replication occurs in the S-phase cell and new genetic material is synthesized as a preparation for the cell division. Synthesis of histones and other DNA associated proteins is markedly increased in the S-phase. The process is highly regulated and requires many different enzymes that include DNA polymerase, primase, ligase, helicase, and topoisomerase. Replication is known to be semiconservative as the original DNA (the parent strand) splits to make a new strand, while retaining the parent strand.[1] 

Replication in prokaryotic and eukaryotic cells is quite similar. The difference in eukaryotic replication lies in the larger amount of DNA that is associated with histones. The prokaryotic DNA is circular and therefore has only one point of origin where replication starts and moves in a bidirectional manner. Eukaryotic cells, on the other hand, have a linear structure that is organized into tight chromosomes around histones.[2][3]

Transcription is the process where a specific segment of DNA is used as a template and copied into an RNA molecule. This synthesis is carried out by an enzyme known as RNA polymerase. The newly synthesized RNA molecule then exits the nucleus and enters the cytoplasm, where it is translated into protein. [4]

Eukaryotic DNA replication occurs in the nucleus of a cell wherein new DNA is made using the original DNA as a template. The process occurs in three stages-initiation, elongation, and termination. Once the DNA is formed, it undergoes the process of transcription synthesizing messenger RNA, which will then be used to generate proteins. Similar to replication, transcription also follows a three-step process of initiation, elongation, and termination. mRNA formation is followed by post-transcriptional modifications in eukaryotic cells and forms the basis of gene expression.

Several issues can occur during replication or transcription, the most common being mutations leading to a loss of function. To avoid these problems, there are enzymes that carry our proofreading and DNA repair as the DNA is being replicated. This ensures high fidelity replication, with very few base pair mismatches. However, problems in proofreading and repair mechanisms can result in mutations, causing the cell to exhibit aberrant behavior. Many mutations may go unnoticed as they are silent or uncovered by the regulatory mechanisms in the cell. Some mutations, however, can directly affect the expression of genes, and cause diseases such as cancer.

Replication

The process of replication is a highly complex process and requires a concerted effort of many different proteins including but not limited to DNA Polymerases, Primase, Helicase, and DNA ligase. In eukaryotes, Polymerases δ and ε are the major replicative enzymes.

DNA has a double helical structure, where the two strands are joined together with hydrogen bonds between the complementary base pairs. Each strand is made up of nucleotides joined together by phosphodiester linkages. For the replication process to begin, the DNA helix must first unwind by removing the hydrogen bonds holding the two strands together. The unwinding process is accomplished by DNA helicase that often starts disconnecting the DNA in a region that is rich in adenine (A) and thymine (T). Since these bases have only two hydrogen bonds, instead of three, it is an ideal place for the helicase to start unwinding the DNA. Unwinding creates a replication fork that has a leading and a lagging strand, a result of the anti-parallel nature of the strands. [5][6][7]

DNA polymerase then starts synthesizing the new strand from 5’ to 3’ direction, but requires a short RNA sequence, approximately 10 nucleotides in length, as a primer to begin the process. An RNA primer is therefore generated with the help of RNA primase enzyme, to initiate placing RNA bases complementary to the template strand bases. The DNA strands are directional with one side (3') denoted by a hydroxyl group and another represented by a phosphate group (5'). Multiple RNA primers are needed for the lagging strand which is then used by the DNA polymerase to begin the elongation phase of DNA replication. DNA replication is high fidelity due to the proofreading ability of DNA polymerase that detects, removes, and fixes any errors made during the replication process. [2][8]

On the leading strand, the DNA polymerase continues in constant motion while the lagging strand has to be copied in short segments in the opposite direction since DNA polymerase only codes in 5' to 3' direction. These short repetitive lagging fragments are known as the "Okazaki fragments". Once the Okazaki fragment synthesis is complete, the RNA primer is no longer required and must be eliminated and replaced with the proper DNA sequence. A flap endonuclease 1 (FEN1) and RNase H remove this RNA primer and the gap is then filled by the action of Polymerase δ that then uses the parental DNA strand as a template to add remaining bases. Finally, another enzyme called DNA ligase connects the Okazaki fragments on the lagging strand by creating the phosphodiester bonds. The generation of these bonds is the final process in DNA replication, resulting in the formation of two new daughter DNA double helices. They are referred to as semiconservative because each double helix now consists of one original parent template strand and a newly synthesized strand.[7][9]

Towards the completion of replication, the newly synthesized lagging strand is shorter with a 3’-overhang. This is thought to be due to the degradation of RNA at the end of the chromosome or inability of primase to lay down a primer at the end. To prevent the loss of genes during successive replications, telomeres are added at the ends of the chromosomes by an enzyme called telomerase. Telomerase enzyme is equipped with both the proteins and RNA that has the ability to base-pair with the 3’-overhang and extend it. Without the telomeres, the chromosomes will gradually shorten with each replication and the cell undergoes premature senescence. [10][11]

Transcription

RNA is synthesized from the DNA template by a process known as transcription. This requires the action of enzymes called RNA polymerases to generate a single-stranded RNA from one of the parent DNA strands. Unlike DNA polymerase, RNA polymerase can initiate RNA synthesis without a primer. While prokaryotes have a single RNA polymerase, eukaryotic cells possess three RNA polymerases. RNA synthesis requires RNA polymerase I for ribosomal RNA, RNA polymerase II for mRNA and microRNA, and RNA polymerase III for tRNA and other small RNAs. Transcription is not as accurate as replication and produces more errors. The replication process is highly accurate, due to the proofreading ability of DNA polymerase.[12]

To initiate the process of transcription, the RNA polymerase must first identify the gene to be transcribed, the correct strand of the dsDNA that it must copy, and bind to a specific sequence on DNA called the promoter. A strand of DNA is read by the RNA polymerase in 3’-5’ direction and its RNA transcript is synthesized in the 5’-3’ direction. The promoter region is known as a TATA box due to the presence of a high frequency of adenine and thymine. In prokaryotes, the TATA box consensus sequence is TATAAT. A similar consensus sequence TATA(A/T)A is present in the eukaryotes. Several other proteins such as transcription factors and binding proteins also participate at the initiation site and function together as a complex.[12][13][13]

Once the RNA polymerase is able to bind to the section of the gene that will undergo transcription, it continues to separate the double helix and synthesize RNA in a 5' to 3' direction. The RNA polymerase places complementary bases to the template strand, except instead of placing thymine with every adenine, the polymerase places a new base called uracil (U). This process is the elongation phase as the RNA polymerase continues down the template creating a new complementary single-stranded RNA. The elongation step proceeds until the polymerase meets a hairpin loop structure known as the termination sequence, which causes the polymerase to fall off, thus beginning the termination phase.

Once synthesized, the RNA undergoes post-transcriptional modification to prevent its degradation during its exit from the nucleus to the cytoplasm where it will be translated to protein. The single-stranded RNA receives a 5' capping by 7-methylguanosine, which is often an mRNA sequence, as well as the addition of poly-A-tail on the 3' end. During these modifications, eukaryotic cells also undergo splicing, in which portions of the RNA called introns are cut out, leaving only the required bases for translation, known as exons. The newly synthesized RNA strand then travel through the pores in the nuclear envelope to the cytosol in order to participate in protein synthesis by the process known as translation.[14][15]