Several types of rRNA are transcribed as a single strand of precursor RNA (Fig. 3-7A). Each rRNA molecule is cleaved following transcription in a process known as trimming. This is a method with finality, since there are cases in which all types of rRNA are needed. Some tRNA molecules are also included in and synthesized from single RNA strands. Like rRNA, several tRNA molecules are synthesized together as one precursor RNA strand and cleaved (Fig. 3-7B). However, one strand does not contain all types of tRNA. One of the enzymes involved in trimming is RNaseP (which contains RNA), and in eubacteria, the enzymatic action is played by RNA rather than by proteins. More detailed trimming processes (not shown in Fig. 3-7) also occur.
RNA Replication and Reverse Transcription
Genes are found in DNA from bacteria to humans, but some viruses, including phages, have single-stranded or double-stranded RNA as a gene carrier. Such viruses have a gene that encodes RNA replicase, and synthesize RNA using RNA as a template after infection. Some viruses that cause cancer in humans and other animals have double-stranded RNA as a gene carrier. Such viruses are called retroviruses; using reverse transcriptase, they reverse-transcribe DNA using RNA as a template after infection. The viral DNA generated is integrated to the DNA of the host cell, replicated with the host’s cellular DNA and passed on to the progeny cells. At the same time, proteins are continuously produced by the oncogene integrated, turning the cell cancerous.
After the formation of an RNA strand, rRNA and tRNA undergo base modification. mRNA also undergoes base modification, but to a lesser extent. The main modification made to rRNA is methylation, in which the methyl group of S-adenosylmethionine is transferred. tRNA receives many types of base modification, and compounds known as minor bases*5 (such as pseudouridine, 4-thiouridine, thymidine, dihydrouridine and 1-methylguanosine) are generated as a result. Minor bases are necessary for tRNA to function. Another important modification type is the enzymatic addition of a three-base sequence, CCA, to the 3’ end of tRNA in eukaryotes. tRNA in prokaryotes has CCA at the 3’ end from the beginning; the 3’ end of tRNA in both eukaryotes and prokaryotes therefore has CCA.
Minor bases: In addition to the five main base types, high molecular DNA and RNA also contain other bases. These are known as minor bases, and are thought to play important functions despite their small quantities.
mRNA in eukaryotes is first transcribed from DNA as pre-mRNA (Fig. 3-8), which becomes complete mRNA after going through the following three main changes (processing):
■Capping (Cap Formation)
A special structure with a phosphate bond between 5’ and 5’ is added to the 5’ end of mRNA. No other nucleotide bonds that form a bond between 5’ and 5’ are known. This is called the cap structure (Fig. 3-9), and is essential when mRNA is used for protein synthesis and binds to ribosomes via special proteins attached to the cap. mRNA in prokaryotes, which have no cap structure, would not function in the protein synthesis apparatus of eukaryotes.
■Addition of Poly-A
A poly-A signal sequence (e.g., AAUAAA) is located near the 3’ end of pre-mRNA, and following enzymatic cleavage at a site approximately 20 bases downstream from the sequence, many As (adenosines) are added to the end. The number of nucleotides added can be from several dozen to thousands. This synthesis does not require a template. Since even mRNA molecules of the same type have different poly-A lengths, the size of the complete mRNA varies. It is suggested that the poly-A strand is necessary for the initiation of protein synthesis and the inhibition of mRNA degradation. In an experiment, mRNA with poly-A can be condensed and purified by attaching it in a complementary fashion to oligo dTs attached to the surface of a fine resin.
The most remarkable part of processing in eukaryotes is splicing. Genes in eukaryotes consist of exons, which contain amino acid sequence information (codes), and introns, which do not, and pre-mRNA containing both exons and introns is first synthesized. In splicing, only introns are removed from the pre-mRNA synthesized, and the exons remaining are connected to form mRNA (Fig. 3-8). To connect two distant exons generated by the removal of introns, a spliceosome - a complex containing non-coding snRNA (small nuclear RNA) - binds near the two breakpoints, pulling them together (Fig. 3-10).
During the process of splicing, some introns may be retained, for example, or two introns and one exon between them may be removed altogether, thereby creating several types of complete mRNA. This mechanism is called alternative splicing.
As a result, several types of protein with different amino acid sequences can be synthesized from such mRNA, each exhibiting different functions. By exploiting this mechanism, one gene can produce several protein types, thus functioning as if it were several genes. This is the reason for the estimation that the number of human genes is virtually 100,000, despite the actual number being 26,000.