Chapter Introduction

RNA Synthesis and Processing

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RNA synthesis is a key step in the expression of genetic information. For eukaryotic cells, the initial RNA transcript (the mRNA precursor) is often spliced, removing introns that do not encode protein sequences. Often, the same pre-mRNA is spliced differently in different cell types or at different developmental stages. In the image at the left, proteins associated with RNA splicing (stained with a fluorescent antibody) highlight regions of the newt genome that are being actively transcribed.

OUTLINE

  1. RNA Polymerases Catalyze Transcription

  2. Transcription in Eukaryotes Is Highly Regulated

  3. The Transcription Products of Eukaryotic Polymerases Are Processed

  4. The Discovery of Catalytic RNA Was Revealing in Regard to Both Mechanism and Evolution

DNA stores genetic information in a stable form that can be readily replicated. The expression of this genetic information requires its flow from DNA to RNA and, usually, to protein, as was introduced in Chapter 4. This chapter examines RNA synthesis, or transcription, which is the process of synthesizing an RNA transcript with the transfer of sequence information from a DNA template. We begin with a discussion of RNA polymerases, the large and complex enzymes that carry out the synthetic process. We then turn to transcription in bacteria and focus on the three stages of transcription: promoter binding and initiation, elongation of the nascent RNA transcript, and termination. We then examine transcription in eukaryotes, focusing on the distinctions between bacterial and eukaryotic transcription.

RNA transcripts in eukaryotes are extensively modified, as exemplified by the capping of the 5′ end of an mRNA precursor and the addition of a long poly(A) tail to its 3′ end. One of the most striking examples of RNA modification is the splicing of mRNA precursors, which is catalyzed by spliceosomes, protein complexes consisting of small nuclear ribonucleoprotein particles (snRNPs). Remarkably, some RNA molecules can splice themselves in the absence of protein. This landmark discovery by Thomas Cech and Sidney Altman revealed that RNA molecules can serve as catalysts, greatly influencing our view of molecular evolution.

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RNA splicing is not merely a curiosity. Many genetic diseases have been associated with mutations that affect RNA splicing. Moreover, the same pre-mRNA can be spliced differently in various cell types, at different stages of development, or in response to other biological signals. In addition, individual bases in some pre-mRNA molecules are changed in a process called RNA editing. One of the biggest surprises of the sequencing of the human genome was that only about 21,000 genes were identified compared with previous estimates of 100,000 or more. The ability of one gene to encode more than one distinct mRNA by alternative splicing and, hence, more than one protein may play a key role in expanding the repertoire of our genomes.

Furthermore, the investigation of different classes of RNA molecules has been one of the most productive areas of biochemical research in recent years. In the next chapter we will explore the long-known ribosomal and transfer RNA molecules that are central to protein synthesis. We have also encountered microRNAs, molecules for which our understanding is still rapidly expanding. Many other classes of RNAs have been discovered more recently, including long non-coding RNAs, the functions of which are still under active investigation.

RNA synthesis comprises three stages: Initiation, elongation, and termination

RNA synthesis is catalyzed by large enzymes called RNA polymerases. The basic biochemistry of RNA synthesis is common to all organisms, commonality that has been beautifully illustrated by the three-dimensional structures of representative RNA polymerases from prokaryotes and eukaryotes (Figure 29.1). Despite substantial differences in size and number of polypeptide subunits, the overall structures of these enzymes are quite similar, revealing a common evolutionary origin.

Figure 29.1: RNA polymerase structures. The three-dimensional structures of RNA polymerases from a prokaryote (Thermus aquaticus) and a eukaryote (Saccharomyces cerevisiae). The two largest subunits for each structure are shown in dark red and dark blue. Notice that both structures contain a central metal ion (green) in their active sites, near a large cleft on the right. The similarity of these structures reveals that these enzymes have the same evolutionary origin and have many mechanistic features in common.
[Drawn from 1I6V.pdb and 1I6H.pdb.]

RNA synthesis, like all biological polymerization reactions, takes place in three stages: initiation, elongation, and termination. RNA polymerases perform multiple functions in this process:

1. They search DNA for initiation sites, also called promoter sites or simply promoters. For instance, E. coli DNA has about 2000 promoter sites in its 4.8 × 106 bp genome.

2. They unwind a short stretch of double-helical DNA to produce single-stranded DNA templates from which the sequence of bases can be easily read out.

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3. They select the correct ribonucleoside triphosphate and catalyze the formation of a phosphodiester bond. This process is repeated many times as the enzyme moves along the DNA template. RNA polymerase is completely processive—a transcript is synthesized from start to end by a single RNA polymerase molecule.

4. They detect termination signals that specify where a transcript ends.

5. They interact with activator and repressor proteins that modulate the rate of transcription initiation over a wide range. Gene expression is controlled substantially at the level of transcription, as will be discussed in detail in Chapters 31 and 32.

The chemistry of RNA synthesis is identical for all forms of RNA, including messenger RNAs, transfer RNAs, ribosomal RNAs, and small regulatory RNAs. The basic steps just outlined apply to all forms. Their synthetic processes differ mainly in regulation, posttranscriptional processing, and the specific RNA polymerase that participates.