Chapter 28:  RNA Synthesis and Splicing

DNA stores genetic information in a stable form that can be readily replicated. However, the expression of this genetic information requires its flow from DNA to RNA to protein, as was introduced in Chapter 5. The present chapter deals with how RNA is synthesized and spliced. We begin with transcription in Escherichia coli and focus on three questions: What are the properties of promoters (the DNA sites at which RNA transcription is initiated), and how do the promoters function? How do RNA polymerase, the DNA template, and the nascent RNA chain interact with one another? How is transcription terminated?

We then turn to transcription in eukaryotes, beginning with promoter structure and the transcription-factor proteins that regulate promoter activity. A distinctive feature of eukaryotic DNA templates is the presence of enhancer sequences that can stimulate transcriptional initiation more than a thousand base pairs away from the start site. Primary 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. Most striking is the splicing of mRNA precursors, which is catalyzed by spliceosomes consisting of small nuclear ribonucleoprotein particles (snRNPs). The small nuclear RNA (snRNA) molecules in these complexes play a key role in directing the alignment of splice sites and in mediating catalysis. Indeed, 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 and greatly influenced our view of molecular evolution.

RNA splicing is not merely a curiosity. Approximately 15% of all genetic diseases are caused by mutations that affect RNA splicing. Morever, 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 approximately 40,000 genes were identified compared with previous estimates of 100,000 or more. The ability of one gene to encode more than one distinct mRNA and, hence, more than one protein may play a key role in expanding the repertoire of our genomes.

28.0.1. An Overview of RNA Synthesis

Figure 28.1

RNA Polymerase Structures

Figure 28.1

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   RNA Polymerase Structures

The three-dimensional structures of RNA polymerases from a prokaryote (Thermus aquaticus) and a eukaryote (Saccharoromyces cerevisiae). The two largest subunits for each structure are shown in dark red and dark blue. The similarity of these structures reveals that these enzymes have the same evolutionary origin and have many mechanistic features in common.

RNA synthesis, or transcription, is the process of transcribing DNA nucleotide sequence information into RNA sequence information. RNA synthesis is catalyzed by a large enzyme called RNA polymerase. The basic biochemistry of RNA synthesis is common to prokaryotes and eukaryotes, although its regulation is more complex in eukaryotes. The close connection between prokaryotic and eukaryotic transcription has been beautifully illustrated by the recently determined three-dimensional structures of representative RNA polymerases from prokaryotes and eukaryotes (Figure 28.1). Despite substantial differences in size and number of polypeptide subunits, the overall structures of these enzymes are quite similar, revealing a common evolutionary origin.

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

• 1

  It searches 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. Because these sequences are on the same molecule of DNA as the genes being transcribed, they are called cis-acting elements.

• 2

  It unwinds a short stretch of double-helical DNA to produce a single-stranded DNA template from which it takes instructions.

• 3

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

• 4

  It detects termination signals that specify where a transcript ends.

• 5

  It interacts with activator and repressor proteins that modulate the rate of transcription initiation over a wide dynamic range. These proteins, which play a more prominent role in eukaryotes than in prokaryotes, are called transcrip-tion factors or trans-acting elements. Gene expression is controlled mainly at the level of transcription, as will be discussed in detail in Chapter 31.

The fundamental reaction of RNA synthesis is the formation of a phosphodiester bond. The 3′-hydroxyl group of the last nucleotide in the chain nucleophilically attacks the α-phosphate group of the incoming nucleoside triphosphate with the concomitant release of a pyrophosphate (see Figure 5.25). This reaction is thermodynamically favorable, and the subsequent degradation of the pyrophosphate to orthophosphate locks the reaction in the direction of RNA synthesis.

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

RNA synthesis is a key step in the expression of genetic (more...)

   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. [(Left) courtesy of Mark B. Roth and Joseph G. Gall.]