8.2Genes Transcription


Types of RNA

There are three main types of RNA (Table 8-1): mRNA, ribosomal RNA (rRNA), and transfer RNA (tRNA). They are all involved in protein synthesis using genetic information (Fig. 8-5). In addition, eukaryotes possess a few small RNA types, including small nuclear RNA (snRNA) (Fig. 8-10) and micro RNA (miRNA) (see Selection 5 of Chapter 10, Fig. 10-10).

Fig. 8-5 Roles of three types of RNA

mRNA carries the genetic information for the primary protein structure contained in DNA to the protein synthesis system. Similar to genes, there are many types of mRNA. mRNA varies widely in size corresponding to the wide range in protein size. It comprises only 1% of the total intracellular RNA content.

Prokaryotes include 5S, 16S, and 23S*1 rRNA (Table 8-1), and eukaryotes include 5S, 5.8S, 18S, and 28S rRNA. Approximately 95% of intracellular RNA is rRNA, which forms complexes called ribosomes with a number of proteins. Ribosomes function as sites for protein synthesis.

*1 The Svedberg unit (S) indicates ultracentrifugation-induced sedimentation velocity. Although proteins with high molecular weights have high S values, no linear relationship exists between molecular weight and the S value, i.e., an S value does not double when molecular weight doubles.

In terms of size, tRNA is 4S, a small RNA of less than 100 bases. A cell has 40–50 types of tRNA, which comprise approximately 5% of all RNA. tRNA plays a role in transportation of amino acids to ribosomes, the site where amino acids are bound together during protein synthesis. Each tRNA is attached to a specific amino acid. For example, the tRNA for phenylalanine and methionine are designated as tRNAPhe and tRNAMet respectively.

In addition, there are numerous types of RNA other than mRNA that contain information to be translated into the primary protein structure, and these are referred to as ncRNA (non-coding RNA). While the characteristics of RNA synthesis regulation vary among RNA types, the basics of the RNA synthesis mechanism are common to all RNA.

Table 8-1 Type of RNA

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Characteristics of Transcription

During DNA synthesis, the entire sequences (DNA) of the parent DNA strand are accurately replicated from end to end and passed from the parent cell to the daughter cell. On the other hand, RNA transcription does not proceed end to end similar to DNA transcription, only the gene segment is transcribed (see Fig. 8-4). Five genes (a–e) in the DNA segment are shown in see Fig. 8-4A, and thus, five types of mRNA are synthesized. Genes c and d show that the other DNA strand is being read in the reverse direction.

Fig. 8-6 Promoter region

A slightly long region is transcribed as RNA, including regions containing amino acid sequence information (coding region) as well as the extra portions on both sides of the regions (see Fig. 8-4B). The promoters shown in see Fig. 8-4 are the sites where RNA polymerase binds to DNA, and thus, they are upstream of all genes (Fig. 8-6).

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Fundamentals of Transcription

During transcription, using one of the DNA double strands as a template, nucleotides are connected one by one to form base pairs with the template bases. However, because U is used in RNA instead of T in DNA, base pairs are produced with U in RNA pairing with A in DNA. The RNA synthesis reaction can be simply described as follows:

[NMP]n + NTP ↔ [NMP]n+1 + PPi

RNA synthesis, same as DNA synthesis, proceeds from 5′ to 3′, which is opposite to the direction of the DNA template, causing the produced RNA strand to run in a direction opposite to the template DNA strand. Although a primer is necessary for DNA synthesis, with reaction impossible when n = 1, a primer is unnecessary for RNA synthesis because initiation is possible when n = 1.

■RNA Polymerase
In E. coli, RNA is synthesized by only one type of RNA polymerase. Eukaryotes have at least three types of RNA polymerase (I, II, and III) with a corresponding division of roles. RNA polymerase I is involved in rRNA synthesis, II in mRNA synthesis, and III in tRNA synthesis. These enzymes are much more complex in structure than prokaryotic enzymes, comprising more than 10 types of subunits.*2

*2 When multiple proteins aggregate and function as a complex, each constituent protein unit is referred to as a subunit. A subunit does not necessarily refer to one individual protein, e.g., a ribosome subunit actually is a complex of RNA and dozens of proteins.

■Upstream and Downstream of Genes and Base Sequence Numbers
The direction toward the part earlier than at the initiation point of transcription is described as upstream of the gene and the direction in which RNA synthesis will proceed is described as downstream of the gene (see Fig. 8-4B). The first base for RNA synthesis is designated as 1 and numbers are assigned to the subsequent bases in the order toward downstream. Conversely, in the direction toward the upstream of a gene (the promoter region), the bases are numbered −1, −2, −3, etc. No base is designated 0. However, while 1 represents the origin of RNA synthesis, the first codon encoding the amino acid of a protein does not correspond to 1 but is located further downstream (with a higher positive number) (Fig. 8-6).

■Binding of Polymerase to the Promoter
Promoter regions in eukaryotes include unique sequences (Fig. 8-6), the TATA box and CCAAT box,*3 which are recognized by general transcription factors*4 (proteins that promote transcription) (see Selection 3 of Chapter 10). Prokaryotes have several types of proteins called σ factors, which promote RNA polymerase binding to a specific promoter. Genetic variation and complexity is much greater in eukaryotes than prokaryotes, with greater variation in promoter sequences and transcription factors that affect them. However, the basic mechanism in eukaryotes and prokaryotes is similar in that both have frequently used basic promoter sequences to which transcription factors bind, thereby recruiting RNA polymerase.

*3 TATA and CCAAT boxes: these sequences are important DNA sequences used when eukaryotic general transcription factors bind to DNA. Some transcription factors recognize the respective sequences of the TATA box (TATAAAA) and CCAAT box (GGCCAATCT). A number of other such recognition sequences are also present.
*4 General transcription factors are proteins (transfactors) needed when RNA polymerase binds to a promoter. These factors bind to specific sequences on promoters, enabling RNA polymerase to bind and initiate RNA synthesis.

■Role of the Promoter and Initiation of Transcription
Promoters are important in determining the position and direction of RNA polymerase action, and they are also important for the initiation of transcription. Because RNA is synthesized in the 5′ to 3′ direction, a DNA strand with the 3′ to 5′ direction in accordance with the direction in which RNA polymerase progresses serves as a template. The transcription factors and RNA polymerase complex bound to DNA unravels or opens the DNA double strands, initiating RNA synthesis.

■Elongation of Transcription
The 5′ end of RNA is pppA or pppG, with a 5′-triphosphate attached to the first nucleotide of the synthesized RNA. The general transcription factors that play a role in RNA polymerase binding do not move along with the enzymes–only RNA polymerase moves along DNA. The RNA strand elongated by the progression of synthesis is released immediately from DNA, while the two unwound DNA strands reform their original double-stranded form on completion of RNA synthesis.

■Termination of Transcription
A terminator is a DNA sequence that indicates transcription termination in prokaryotes. Terminated RNA is known to exhibit a structure when dissociated from DNA. The synthesized RNA forms a double-stranded structure with itself (hairpin structure) and detaches from the DNA template. The transcription termination mechanism in eukaryotes is not well understood.

■Gene Amplification in rRNA and tRNA
The number of functioning (transcribed) genes in E. coli is over 2000, and this figure is believed to be much higher in humans. Based on the mRNA information transcribed from those genes, the gene products (proteins) are all synthesized by ribosomes. Because protein synthesis systems are shared, they must exist in large amounts to handle copious RNA translation from all types of genes. Thus, rRNA and tRNA that function at the sites of protein synthesis must be synthesized in large amounts for which their genes must be present in large numbers (moderately repetitive sequences) in addition to their transcription being actively performed. The same gene existing in large quantities is evidence of gene amplification./p>


RNA Synthesis Inhibitors

Antibiotics and toxins that inhibit transcription are used as pharmaceutical products and research reagents. Rifampicin is an antibiotic that inhibits transcription in prokaryotes. Actinomycin D is an antibiotic that acts as a transcriptional inhibitor in prokaryotes and eukaryotes. It has a three-membered ring that intercalates between GC base pairs in DNA. α-Amanitin is a cyclic peptide toxin derived from Amanita pantherina. It inhibits eukaryotic RNA polymerase II at low concentrations. It also inhibits RNA polymerase III at high concentrations, although it never inhibits RNA polymerase I. Cordycepin is incorporated as A in RNA synthesis, stopping elongation of the strand because of the presence of a deoxy group in the 3′ position. It is especially an inhibitor of eukaryotic mRNA synthesis because it inhibits poly(A) addition reactions.


RNA Replication and Reverse Transcription

Genes exist as DNA in both bacteria and humans, but genes in viruses or phages may exist as single- or double-stranded RNA. These viruses contain genes for RNA replicase, and after infection, they use RNA as the template for RNA synthesis. Some oncoviruses that cause cancer in animals and humans contain genes in the form of double-stranded RNA. These types of viruses are called retroviruses. They contain reverse transcriptase to transcribe DNA in the reverse direction using genetic RNA as a template after infection. This viral DNA is then incorporated into cellular DNA and replicated and transmitted to daughter cells in the same manner as normal cellular DNA. Cancer proteins produced from oncogenes lead cells to a cancerous state.


Reverse Transcriptase and cDNA

Reverse transcriptase is an enzyme that uses RNA as a template to synthesize DNA. A DNA strand complementary to the mRNA sequence (template) can be synthesized by reverse transcriptase in the presence of an appropriate primer. Double-stranded DNA can be synthesized by DNA synthetase using that DNA as a template. DNA produced in this manner using RNA as a template is called complementary DNA (cDNA). With regard to eukaryotes, the difference between cDNA and genomic DNA is that cDNA lacks intron regions. RNA transcribed from cDNA has the same amino acid sequence as mRNA transcribed from genomic DNA.
Compared with genomic DNA, cDNA is easier to handle because of its small size, and thus, it has a high utility value. cDNA is commonly used in research in the following manner: cDNA or cDNA with various mutations can be bound to an appropriate promoter DNA and introduced into cells. By observing changes in the form and function of these cells, the function of proteins synthesized through transcription and translation can be examined. If the introduced cDNA is stably incorporated into cellular DNA, properties differing from those of the original cell can be maintained in the obtained transformed cells. In addition to broad basic research, transgenic animals and plants produced with these cells are used in the fields of animal husbandry and agriculture, respectively. One example is the genetically modified organisms (GMOs).
Another example is the large amounts of useful products produced by introducing human cDNAs into Escherichia coli.

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