Biochemistry - The Chemical Reactions of Living Cells, Volume 3 - D. Metzler 1980

Biochemical Genetics and the Synthesis of Nucleic Acids and Proteins
Transcription of RNA Molecules
Transcription in Eukaryotic Cells

In Cells with a true membrane-bound Nucleus, Messenger RNA molecules have a relatively long lifespan. Over the course of their lifetime, they must exit The Nucleus into the Cytoplasm and make their way to the sites of Protein Synthesis. Aside from the obvious necessity of surviving longer and traveling greater distances compared to bacterial mRNA, eukaryotic mRNAs differ from them in A number of other parameters that are not yet fully understood. It is quite possible, for example, that in eukaryotes, mRNA molecules are transcribed from individual genes, and polygenic operons do not typically function in these cells. A new type of RNA, designated as heterogeneous nuclear RNA (hnRNA), has been discovered in Eukaryotic cells. It has been hypothesized that this RNA, which accounts for the bulk of nuclear RNA, serves as the precursor to mRNA. Much like mRNA, it is similar in nucleotide composition to DNA. The Molecular Weight of this RNA typically ranges from 10⁵ to 2∙10⁷ (corresponding to 1,500—30,000 NUCLEOTIDES). hnRNA molecules are characterized by a capacity for rapid turnover—for the majority of them, the half-life does not exceed 10 min. However, some hnRNA species can persist for up to 20 h. Strikingly, only about 10% of hnRNA ever leaves the nucleus, while the greater part of it is degraded within the nucleus without ever passing into the cytoplasm [77—79].

An unexpected discovery helped confirm that hnRNA is indeed the precursor to mRNA. It was shown that both hnRNA and eukaryotic mRNA possess long chains of polyadenylic acid [poly(A)] at their 3'-ends. (An apparent exception is found in mRNA molecules that encode the synthesis of Histones—the major Nuclear Proteins found in eukaryotic cells.) Typically, about 200 adenylic acid residues are added to the end of the hnRNA chain, seemingly through the action of a specific enzyme that operates post-transcriptionally [79a].

Evidence indicates that the mRNA arriving in the cytoplasm—having been generated by the Cleavage of hnRNA—still contains 50—75 adenylic acid residues. These residues are gradually (though seemingly not completely) cleaved from the mRNA. What is the function of such poly(A) tracts? We do not yet know the answer. According to one hypothesis, poly(A) sequences are required in some capacity for The transport of mRNA out of the nucleus. Another hypothesis suggests that after each round of Translation, as the mRNA dissociates from the ribosome, one or more nucleotides are cleaved from its 3'-end. Thus, in this view, the individual units of the 3'-terminal poly(A) tracts act as "tickets," each entitling the molecule to one "trip" through the ribosome. The depletion of all A residues serves as a signal for The Cell to degrade the mRNA. Although the presence of poly(A) segments in mRNA is considered a hallmark of eukaryotic cells, rapidly turning over poly(A) sequences have also been discovered in E. coli [80].

Another striking feature of mRNA, discovered relatively recently, is that the 5'-ends of many mRNA molecules in both eukaryotes and Viruses are capped with specialized terminal structures containing 7-methylguanosine in the form of a zwitterion generated by proton abstraction [81—83]. Note the triphosphate bridge linking 7-methyladenosine to the mRNA. It can be formed in the following manner [82]. The 5'-end of the initial RNA transcript bears a triphosphate group because initiation is primed by a nucleoside triphosphate. One of these phosphate groups can be cleaved off, leaving a diphosphate that then reacts with GTP. Subsequently, the 7-methyl group, as well as the 2'-methyl group of the ribose moiety¹⁾ located at the 5'-end of the polynucleotide, is transferred from S-adenosylmethionine with the aid of a specific methylase.

Class="center">

Poliovirus mRNA is unusual in that it remains uncapped in infected cells [83a].

The Nucleolus

Through numerous studies, it has been conclusively established that the nucleolus is the site of ribosomal RNA Synthesis (a nucleus may contain one or several nucleoli). Eukaryotic Ribosomes contain four Different types of RNA molecules, the sizes of which are listed below.

¹⁾ The adjacent ribose residue is also occasionally methylated, and N⁶-methyladenosine may be located at the 3'-end adjacent to the poly(A) tract [83].

The 28S, 18S, and 5.8S molecules are derived from a high-molecular-weight (4∙10⁶) 45S pre-rRNA. This precursor is transcribed within the central region of the nucleolus. As the pre-rRNA molecule moves out into the outer "cortex," it undergoes stepwise cleavage [equation (15-6)] [57, 84, 85].

Electron Microscopy has provided direct confirmation of the linkage between precursor molecules (Fig. 15-11) [85a, b]. It is worth noting that the 18S subunit of the 45S RNA lies closest to the 5'-end, exactly like the 16S rRNA in the large prokaryotic rRNA transcript (Section B,3). It is possible that the number of Processing steps is actually greater than shown in the figure. For instance, The formation of the 45S RNA may be preceded by 46S and 47S molecules.

FIG. 15-11. A. Electron micrograph of the 45S rRNA precursor from HeLa cells after molecular spreading in an 80% formamide and 4 M urea solution. The molecule was visualized using negative staining. B. Outline tracing of the molecule shown in A. Several regions with double-stranded hairpin secondary structures are visible. Regions corresponding to 28S and 18S rRNAs are indicated. C. 32S rRNA. D. 28S rRNA. Note that the same secondary structures seen in the 32S and 45S precursors are also discernible in the 28S rRNA [85a].

FIG. 15-12. Nucleotide sequence of human 5S rRNA (KB carcinoma cells). A and B. Two possible structural Conformations (Sirlin J. L., "Biology of RNA," p. 67, Academic Press, New York, 1973). C. Alternative base-pairing schemes in eukaryotic 5S rRNA (top) and prokaryotic 5S rRNA (bottom). Letters inside loops designate nucleosides conserved across human KB cells, X. laevis, and T. utilis 5S rRNAs (top), and across E. coli, P. fluorescens, and B. stearothermophilus 5S rRNAs (bottom). Boxed nucleotides are found at the corresponding positions in both eukaryotic and prokaryotic 5S rRNAs (Nishikawa K., Takemura S., J. Biochem., Tokyo, 76: 935—947, 1974).

Direct visualization of the disordered central zone of nucleoli using electron microscopy has played a critically important role in The Study of the nucleolus [59, 86]. Nascent protein-coated RNA strands can be seen emerging from the DNA templates of pre-RNA genes (Fig. 15-7). Approximately 80 to 100 RNA strands of varying lengths are transcribed simultaneously from a single Gene. According to electron microscopic data, the total length of the gene is 2.3 µm, which is only slightly less than the calculated length of a fully extended DNA molecule (in the B-form). However, judging by the length of the transcripts being produced, the pre-rRNA chains are folded multiple times to form a compact Structure.

In most organisms, the haploid gene set contains typically only a single copy of a given gene type. Ribosomal RNA genes, however, are represented in The Genome by multiple copies. For instance, 130 to 190 copies of the 45S rRNA gene are found in Drosophila.

The eukaryotic 5S rRNA gene is unlinked to the 45S rRNA gene and is localized outside the nucleolus. In Drosophila, approximately 500 copies of the 5S rRNA gene are located on the right arm of chromosome 2. RNA polymerase III is responsible for the synthesis of 5S rRNA (The nucleotide sequence of which is shown in Fig. 15-12). A characteristic feature of 5S rRNA is its ability to fold into alternative conformations, and it remains unclear how it is packaged within ribosomes [87].