RNA polymerases in eukaryote

The primary transcripts generated by RNA polymerase II—one of three distinct nuclear DNA-depen-dent RNA polymerases in eukaryotes—are promptly capped by 7-methylguanosine triphosphate caps (Figure 35-10) that persist and eventually appear on the 5 end of mature cytoplasmic mRNA. These caps are necessary for the subsequent processing of the primary transcript to mRNA, for the translation of the mRNA, and for protection of the mRNA against exonucleolytic attack. 

With respect to the general mechanism of RNA chain growth, are there any differences among the various types of RNA polymerase in eukaryotic cells ... 

General transcription factors belong to a group of proteins which assemble at the TATA box or a similar region in the promoter.They are required for the initiation of transcription by the DNA-dependent RNA polymerase in eukaryotic cells. 

Rifampicin (Fig. 10.70) is a semisynthetic rifamycin made from rifamycin B—an antibiotic isolated from Streptomyces mediterranei. It inhibits Gram-positive bacteria and works by binding non-covalently to RNA polymerase and inhibiting RNA synthesis. The DNA-dependent RNA polymerases in eukaryotic cells are unaffected, since the drug binds to a peptide chain not present in the mammalian RNA polymerase. It is therefore highly selective. 

There are three RNA polymerases in eukaryotes, of which Pol II produces mRNA. 

RNA polymerase II the RNA polymerase in eukaryotes that makes mRNA also called RNA polymerase B (11.4) rRNA (ribosomal RNA) the kind of RNA found in ribosomes (9.5)... 

There are three RNA polymerases in eukaryotes, compared with one in prokaryotes. There are many more transcription factors in eukaryotes, including complexes of them necessary for polymerase recruitment. RNA is extensively processed after transcription in eukaryotes, and, in most cases, the mRNA must leave the nucleus to be translated, whereas translation and transcription can occur at the same time in prokaryotes. 

Roeder, R.G., Rutter, W.J. Multiple forms of DNA-dependent RNA polymerase in eukaryotic organisms. Nature (Lond.) 224,234-237 (1969)... 

The elongation of RNA strands by RNA polymerase in both bacteria and eukaryotes is inhibited by the antibiotic actinomycin D (Fig. 26-10). The planar portion of this molecule inserts (intercalates) into the doublehelical DNA between successive G=C base pairs, deforming the DNA. This prevents movement of the polymerase along the template. Because actinomycin D... 

There are three distinct classes of RNA polymerase in the nucleus of eukaryotic cells. All are large enzymes with multiple subunits. Each class of RNA polymerase recognizes particular types of genes. 

DNA are readily accessible to RNA polymerase binding and transcription. By contrast, most DNA in eukaryotic cells exists in a condensed form (chromatin), which is not readily accessible to transcription. The small fraction of DNA accessible to the RNA polymerase in any given cell type is especially sensitive to cleavage by mild treatment with bovine pancreatic DNase I. These regions of the DNA often contain bound RNA polymerase, modified histones, and additional nonhistone proteins. Active regions are often undermethylated compared with the total DNA. Most of the methylated groups in DNA are on the C residues in the CG sequence. 

The mRNA required for in vitro translation can itself be produced by in vitro synthesis. Commercially available kits allow DNA cloned downstream of T7-, T3 or SP6-promoters to be transcribed effectively in vitro by the relevant RNA polymerases. In coupled transcription-translation, it should be remembered that translation of eukaryotic mRNA requires a 5 cap upstream of the initiation codon, and similarly, for prokaryotic translation there should be an appropriately positioned ribosome binding site. Commercial kits are also available for combined in vitro transcription and translation. 

RNA synthesis in eukaryotes takes place in the nucleus, whereas protein synthesis takes place in the cytoplasm. There are three types of RNA polymerase in the nucleus RNA polymerase I makes ribosomal RNA precursors, II makes messenger RNA precursors, and III makes transfer RNA precursors. Eukaryotic promoters are complex, being composed... 

RNA polymerases, the eukaryotic process in an unusual configuration called a lariat. Splicing takes place within a spliceo-... 

In prokaryotic cells, which have no nuclei, translation of an mRNA into protein can begin from the 5 end of the mRNA even while the 3 end is still being synthesized by RNA polymerase. In other words, transcription and translation can occur concurrently in prokaryotes. In eukaryotic cells, however, not only is the nucleus separated from the cytoplasm where translation occurs, but also the primary transcripts of protein-coding genes are precursor mRNAs (pre-mRNAs) that must undergo several modifications, collectively termed RNA processing, to yield a functional mRNA (see Figure... 

Polymerase a was the first discovered, and it has the most subunits. It also has the ability to make primers, but it lacks a 3 5 proofreading activity and has low processivity. After making the RNA primer, Pol a adds about 20 nucleotides and is then replaced by Pol 5 and e. Polymerase 5 is the principal DNA polymerase in eukaryotes. It interacts with a special protein called PCNA ior proliferating cell nuclear antigen). PCNA is the eukaryotic equivalent of the part of Pol III that functions as a sliding clamp (P). It is a trimer of three identical proteins that surround the DNA (Figure 10.19). The role of DNA polymerase e is less clear. It may replace polymerase 5 in lagging strand synthesis. DNA polymerase p appears to be a repair enzyme. DNA polymerase y carries out DNA replication in mitochondria. Several... 

To make any particular RNA product, the RNA polymerase reads one of the DNA strands, called the template strand. It moves along the template strand from 3 to 5 and produces the RNA from 5 to 3. The other strand of DNA is called the coding strand, and its sequence matches that of the RNA produced. In eukaryotes, the opposite strand is often used to produce small noncoding RNAs their function in gene expression is being actively studied. 

Like prokaryotic RNA polymerase, each eukaryotic enzyme copies DNA from the 3 end, thus catalyzing mRNA formation in the 5 => 3 direction and synthesizing RNA complementary to the antisense DNA template strand. The reaction requires the precursor nucleotides ATP, GTP, CTP, and UTP and does not require a primer for transcription initiation. Unlike the prokaryotic bacterial polymerases, the eukaryotic RNA polymerases require the presence of additional initiation proteins before they are able to bind to promoters and initiate transcription. The five stages of eukaryotic transcription include initiation, elongation and termination, capping, polyadenylation, and splicing. 

Outline RNA processing in eukaryotes. Name the alterations made to the RNA after it is initially formed by RNA polymerase. 

Match the descriptions in the right column with the appropriate eukaryotic DNA-dependent RNA polymerases in the left column. 

Prokaryotic and eukaryotic RNA transcriptions show strong parallels though there are several important differences. A major distinction between prokaryotes and eukaryotes is the move from one prokaryotic enzyme that can faithfully transcribe DNA into RNA to three eukaryotic RNA polymerases. The eukaryotic RNA transcripts are precursors (e.g. pre-mRNA, pre-rRNA and pre-tRNA), which undergo processing to form respective mature RNAs. Furthermore, eukaryotic mRNAs are polyadenylated. A database for mammalian mRNA polyadenylation is available at PolyA DB (http // polya.umdnj.edu/polyadb). The eukaryotic transcription is tightly regulated and various proteins/factors known as transcription factors (TF) are involved in the eukaryotic transcription. The classification of transcription factors can be found at TRANFAC (http //transfac.gbf.de/TRANFAC/cl/cl.html). 

The mitochondrial RNA polymerase from diflEerent sources has been solubilized and partially purified in recent years. The few data available on the structure indicate that the mitochondrial RNA polymerase has very unusual properties compared with those of other RNA polymerases, either eukaryotic or bacterial. Bacterial RNA polymerases, and eukaryotic nuclear polymerases, consist of several typical subunits assembled to pve a molecular weight of about half a million. However, for mitochondrial RNA polymerases molecular weights of 46,000 Xenopus, Wu and Dawid, 1972) and of 64,000 (Kiintzel and Schafer, 1971) have been published. This corresponds to the size of the smaller subunits from other well-known RNA polymerases. Further studies are clearly needed. 

RNA polymerase has now been purified in various laboratories from various mammalian sources [188], and RNA polymerase of eukaryotic nuclei always exists in multiple forms that can be distinguished by the cation requirement, their sensitivity to a-amanitin, and their ability to react with specific templates. One enzyme (polymerase I) is found in the nucleolus, the others in the nucleoplasm. All mammalian ribonucle-ases are complex proteins formed of several subunits. Three types of RNA polymerases have been purified from ascites tumor cells by chromatography on car-boxymethyl-cellulose. Two are nucleolar and one is nucleoplasmic. Protein factors of unknown nature that stimulate all three enzymes have been found in calf thymus, rat liver, and ascites cells. These factors can be separated into two classes heat stable and heat labile. Both types stimulate the activity of the Novikoff RNA polymerase several-fold, but only with native DNA as templates. The factors have no effect on E. coli RNA polymerase [266-267]. For further information, refer to the review of Jacobs [189]. 

RNA Polymerase I. Eukaryotic cells contain several hundred copies of the genes for ribosomal RNA. Each unit encodes the sequences for 28S, 18S, and 5.8S RNA, which are transcribed, together with transcribed spacer sequences, into a single precursor molecule. Transcription units alternate with non-transcribed spacers and it is these regions that have been searched for the promoter regions for RNA polymerase I. The available data to date cannot, however, be said to produce a clear picture and this is aggravated by the absence of a reliable in vitro system. 

Roeder, R. G., 1976, Eukaryotic nuclear RNA polymerases, in RNA Polymerase (R. Losick and M. Chamberlin, eds.), pp. 285-329, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. 

As mentioned earlier, RNA synthesis is catalyzed by the RNA polymerase in all organisms. Prokaryotes express a single RNA polymerase used for synthesis of all RNAs, while eukaryotes encode multiple RNA polymerases with dedicated functions. RNA polymerase I (Pol I) in eukaryotic cells is responsible for synthesis of ribosomal RNA, which accounts for more than 70% of total RNA in the cell. Pol III catalyzes synthesis of small RNA molecules, including transfer RNAs which bring in appropriate amino acids to the ribosome for protein synthesis by using their anti-codon triplet bases. Pol II is responsible for synthesis of all other RNA, specifically mRNA. 

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