The transcription process

The initiator nucleotide binds to the complex and the first phos-phodiester bonds are made, accompanied by release of o. The remaining core polymerase is now in the elongation mode. Several experimental observations support the picture presented in the next figure, namely the fact that less than one a exists in the cell per core enzyme in each cell. 

Elongation is the function of the RNA polymerase core enzyme. RNA polymerase moves along the template, locally unzipping the DNA double helix. This allows a transient base pairing between the incoming nucleotide and newly-synthesized RNA and the DNA template strand. As it is made, the RNA transcript forms secondary structure 

Rho factor A protein that recognizes terminator regions. 

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One way to control how much of something a cell uses or makes is to control the levels of the enzymes that are required to metabolize it (Fig. 5-7). Whether or not transcription happens is controlled by the binding of specific proteins to the DNA. When they bind to DNA, these proteins can either help or hinder the transcription process. Positive and negative refer only to the effect a protein has when it binds to the DNA. A positive effect is when the protein binds to the DNA and turns on the transcription of the gene. A negative effect is when the binding of the protein to the DNA turns off transcription. 

These three compounds exert many similar effects in nucleotide metabolism of chicks and rats [167]. They cause an increase of the liver RNA content and of the nucleotide content of the acid-soluble fraction in chicks [168], as well as an increase in rate of turnover of these polynucleotide structures [169,170]. Further experiments in chicks indicate that orotic acid, vitamin B12 and methionine exert a certain action on the activity of liver deoxyribonuclease, but have no effect on ribonuclease. Their effect is believed to be on the biosynthetic process rather than on catabolism [171]. Both orotic acid and vitamin Bu increase the levels of dihydrofolate reductase (EC 1.5.1.4), formyltetrahydrofolate synthetase and serine hydroxymethyl transferase in the chicken liver when added in diet. It is believed that orotic acid may act directly on the enzymes involved in the synthesis and interconversion of one-carbon folic acid derivatives [172]. The protein incorporation of serine, but not of leucine or methionine, is increased in the presence of either orotic acid or vitamin B12 [173]. In addition, these two compounds also exert a similar effect on the increased formate incorporation into the RNA of liver cell fractions in chicks [174—176]. It is therefore postulated that there may be a common role of orotic acid and vitamin Bj2 at the level of the transcription process in m-RNA biosynthesis [174—176]. 

For the genetic information stored in DNA to become effective, it has to be rewritten (transcribed) into RNA. DNA only serves as a template and is not altered in any way by the transcription process. Transcribable segments of DNA that code for a defined product are called genes. It is estimated that the mammalian genome contains 30 000-40 000 genes, which together account for less than 5% of the DNA. 

The large subimit of RNA polymerase II plays an important role at the beginning of the transcription process. The large subimit of the mammalian enzyme contains 52 copies of the heptamer sequence YSPTSPS in the C-terminal domain (CTD) at which phosphorylation occurs. Phosphorylation occurs extensively on the Ser-residues of the CTD, to a lesser degree at the Thr-residues, and, very rarely, at the Tyr-residues. Two forms of RNA polymerase II can be isolated from cellular extracts a underphosphory-lated form and a hyper-phosphorylated form. The isoforms fulfill different functions RNA polymerase found in the initiation complex tends to display little or no phosphorylation at the C-terminus of the large subunit, while RNA polymerase II active in elongation is hyperphosphorylated in this region of the protein. 

An example for how protein phosphorylation can influence the transcription process is the transition from the initiation to the elongation process for RNA polymerase II (see 1.4.2.4). 

Dining elongation the RNA polymerase is capable of displacing the nucleosome. The histone octamer does not dissociate completely from the DNA. The nucleosome is thus a mobile entity that plays a dynamic role in the transcription process. 

Unlike DNA polymerase, RNA polymerase does not require a primer to initiate synthesis. Initiation occurs when RNA polymerase binds at specific DNA sequences called promoters (described below). The 5 -triphos-phate group of the first residue in a nascent (newly formed) RNA molecule is not cleaved to release PPj, but instead remains intact throughout the transcription process. During the elongation phase of transcription, the growing end of the new RNA strand base-pairs temporarily with the DNA template to form a short hybrid... 

Shortly after Ochoa s studies, Sam Weiss began a search for a DNA-directed RNA polymerase. His experimental design was influenced by the theory that RNA must be made on a DNA template if it is to carry the genetic message. He was also influenced by Komberg s discovery that DNA synthesis required nucleoside triphosphates for substrates rather than nucleoside diphosphates. With crude liver extracts, Weiss was able to demonstrate a capacity for RNA synthesis that was severely inhibited by DNase. Weiss s results touched off systematic investigations of RNA metabolism in many laboratories. A continuous expansion of research effort from that time on has yielded a wealth of understanding about the transcription process and related aspects of RNA metabolism. 

All tRNAs contain several ribonucleotides that differ from the usual four (12 in the case of tRNAphe). The structures for some of these are shown in figure 28.4. Only four ribonucleotides are incorporated into RNA in the transcription process. All of the rare bases found in the mature tRNA result from posttranscriptional modification. 

PolIII polymerase also transcribes tRNA genes. In this case TFIIIA does not participate in the transcription process (see fig. 28.12c). 

In the transcription process the two DNA strands are separated and the antisense DNA strand paired with its complementary RNA bases by enzymes called RNA polymerases to produce mRNA that encodes the same sequence of bases as the sense DNA strand. The only difference between the DNA and RNA sequences is that the saccharide section of the nucleoside is ribose rather than deoxyribose and uracil takes the place of thymine. The effects of these changes are that the hydrophobic 5-methyl group of thymine has been removed to generate uracil and a 2 -hydroxy group is present in the linking saccharide (Table 2.4). 

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