Eukaryotic mRNA synthesis

Eukaryotic mRNA synthesis results in a pre-mRNA precursor that contains extensive amounts of excess RNA (introns) that must be precisely removed by RNA splicing to generate functional, translatable mRNA composed of exonic coding and noncoding sequences. 

Important pathways requiring SAM include synthesis of epinephrine and of the 7-methylgua-nine cap on eukaryotic mRNA, Synthesis of SAM from methionine is shown in Figure T17-3. After donating the methyl group, SAM is converted to homocysteine and remethylated in a reaction catalyzed by N-methyl THF-homocysteine methyltransferase requirii both vitamin Bj2 and N-meth d-THF. The methionine produced is once again used to make SAM. 

A third alternative starts with an extract of RNA, not DNA. Mature eukaryotic mRNA contains a long run or tail of adenine residues at its 3 end. The poly(rA) tail can be hybridized with an oligomer of thymine residues, and the oligo(dT) can then be used as a primer for a particular kind of DNA polymerase known as reverse transcriptase. This enzyme, a polymerase associated with retroviruses, will use RNA as a template to make a complementary DNA copy of the RNA, creating a DNA-RNA double-stranded hybrid. In another round of synthesis, the enzyme can replace the RNA strand entirely with DNA, so that the RNA-DNA hybrid is completely converted to double-stranded DNA containing an exact copy of the original RNA sequence. This DNA molecule is known as cDNA because it has a strand that is complementary to (or a copy of) the original RNA. 

RNA-Dependent Synthesis of RNA and DNA Updates coverage on mechanisms of mRNA processing Adds a subsection on the 5 cap of eukaryotic mRNAs... 

The pathway of protein synthesis translates the three-letter alphabet of nucleotide sequences on mRNA into the twenty-letter alphabet of amino acids that constitute proteins. The mRNA is translated from its 5 -end to its 3 -end, producing a protein synthesized from its amino-terminal end to its carboxyl-terminal end. Prokaryotic mRNAs often have several coding regions, that is, they are polycistronic (see p. 420). Each coding region has its own initiation codon and produces a separate species of polypeptide. In contrast, each eukaryotic mRNA codes for only one polypeptide chain, that is, it is monocistronic. The process of translation is divided into three separate steps initiation, elongation, and termination. The polypeptide chains produced may be modified by posttranslational modification. Eukaryotic protein synthesis resembles that of prokaryotes in most details. [Note Individual differences are mentioned in the text.]... 

Zhao, J., Hyman, L. and Moore, C. (1999) Formation of mRNA 3 ends in eukaryotes mechanism, regulation, and interrelationships with other steps in mRNA synthesis. Microbiol. Mol. Biol. Rev., 63, 405 145. 

In at least one eukaryote, Tetmhymem, the pre-rRNA molecule contains an intron. Removal of the intron during processing of the pre-rRNA does not require the assistance of any protein Instead, in the presence of guanosine, GMP, GDP or GTP, the intron excises itself, a phenomenon known as selfsplicing. This was the first demonstration of ribozymes, that is, catalytic RNA molecules that catalyze specific reactions. The list of ribozymes is growing. For example, self-splicing introns have been discovered in some eukaryotic mRNAs and even peptidyl transferase, a key enzyme activity in protein synthesis, is now known to be a ribozyme (see Topic H2). 

Both prokaryotes and eukaryotes initiate protein synthesis with a specialized methionyl-tRNA in response to an AUG initiation codon. Eukaryotes, however, use an initiator met—tRNAmeti—that is not formylated. Recognition of the initiator AUG is also different. Only one coding sequence exists per eukaryotic mRNA, and eukaryotic mRNAs are capped. Initiation, therefore, uses a specialized capbinding initiation factor to position the mRNA on the small riboso-mal subunit. Usually, the first AUG after the cap (that is, 3 to it) is used for initiation. 

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. 

Figure 28.15. Transcription and Translation. These two processes are closely coupled in prokaryotes, whereas they are spacially and temporally separate in eukaryotes. (A) In prokaryotes, the primary transcript serves as mRNA and is used immediately as the template for protein synthesis. (B) In eukaryotes, mRNA precursors are processed and spliced in the nucleus before being transported to the cytosol for translation into protein. [After J. Darnell, H. Lodish, and D. Baltimore. Molecular Cell Biology, 2d ed. (Scientific American Books, 1990), p. 230.]...

Fig. 11.8 Schematic presentation of mRNA synthesis in Prokaryotes Eukaryotic RNA Polymerases...

C. Synthesis of RNA in nuclei of eukaryotes 1. mRNA synthesis (Figure 3-19)... 

Only ribonucleoside 5 -triphosphates participate in RNA synthesis, and the first base to be laid down in the initiation event is a triphosphate. Its 3 -OH group is the point of attachment of the subsequent nucleotide. Thus, the 5 end of a growing RNA molecule terminates with a triphosphate. In tRNAs and rRNAs, and in eukaryotic mRNAs, the triphosphate group is removed. 

In prokaryotes, mRNA synthesis can be controlled simply by regulating initiation of transcription. In eukaryotes, mRNA is formed from a primary transcript followed by a series of processing events (e.g., intron excision, polyadenylylation). Eukaryotes regulate not only transcription initiation but the various stages of processing as well. 

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