DNA-Dependent Synthesis of RNA:- RNA Is Synthesized by RNA Polymerases
The discovery of DNA polymerase and its dependence on a DNA template spurred a search for an enzyme that synthesizes RNA complementary to a DNA strand. By 1960, four research groups had independently detected an enzyme in cellular extracts that could form an RNA polymer from ribonucleoside 5-triphosphates. Subsequent work on the purified Escherichia coli RNA polymerase helped to define the fundamental properties of transcription (Fig. 1). DNA-dependent RNA polymerase requires, in addition to a DNA template, all four ribonucleoside 5-triphosphates (ATP, GTP, UTP, and CTP) as precursors of the nucleotide units of RNA, as well as Mg2+. The protein also binds one Zn2+. The chemistry and mechanism of RNA synthesis closely re semble those used by DNA polymerases . RNA polymerase elongates an RNA strand by adding ribonucleotide units to the 3-hydroxyl end, building RNA in the 5n3 direction. The 3-hydroxyl group acts as a nucleophile, attacking the phosphate of the incoming ribonucleoside triphosphate (Fig. 1b) and releasing pyrophosphate. The overall reaction is
RNA polymerase requires DNA for activity and is most active when bound to a double-stranded DNA. As noted above, only one of the two DNA strands serves as a template. The template DNA strand is copied in the 3→5 direction (antiparallel to the new RNA strand), just as in DNA replication. Each nucleotide in the newly formed RNA is selected by Watson-Crick base-pairing interactions; U residues are inserted in the RNA to pair with A residues in the DNA template, G residues are inserted to pair with C residues, and so on. Base-pair geometry may also play a role in base selection. Unlike DNA polymerase, RNA polymerase does not require a primer to initiate synthesis. Initiation occurs when RNA polymerase binds at specific DNA sequences called promoters (described below). The 5-triphosphate group of the first residue in a nascent (newly formed) RNA molecule is not cleaved to release PPi, but instead remains intact throughout the transcription process. During the elongation phase of transcription, the growing end of the new RNA strand base-pairs temporarily with the DNA template to form a short hybrid (b) Catalytic mechanism of RNA synthesis by RNA polymerase. Note that this is essentially the same mechanism used by DNA poly RNA-DNA double helix, estimated to be 8 bp long (Fig. 1a). The RNA in this hybrid duplex “peels off” shortly after its formation, and the DNA duplex re-forms.
MECHANISM FIGURE 1 Transcription by RNA polymerase in E. coli. For synthesis of an RNA strand complementary to one of two DNA strands in a double helix, the DNA is transiently unwound. (a) About 17 bp are unwound at any given time. RNA polymerase and the bound transcription bubble move from left to right along the DNA as shown; facilitating RNA synthesis. The DNA is unwound ahead and rewound behind as RNA is transcribed. Red arrows show the direction in which the DNA must rotate to permit this process. As the DNA is rewound, the RNA-DNA hybrid is displaced and the RNA strand extruded. The RNA polymerase is in close contact with the DNA ahead of the transcription bubble, as well as with the separated DNA strands and the RNA within and immediately behind the bubble. A channel in the protein funnels new nucleoside triphosphates (NTPs) to the polymerase active site. The polymerase footprint encompasses about 35 bp of DNA during elongation.
To enable RNA polymerase to synthesize an RNA strand complementary to one of the DNA strands, the DNA duplex must unwind over a short distance, forming a transcription “bubble.” During transcription, the E. coli RNA polymerase generally keeps about 17 bp unwound. The 8 bp RNA-DNA hybrid occurs in this un wound region. Elongation of a transcript by E. coli RNA polymerase proceeds at a rate of 50 to 90 nucleotides/s. Because DNA is a helix, movement of a transcription bubble requires considerable strand rotation of the nucleic acid molecules. DNA strand rotation is restricted in most DNAs by DNA-binding proteins and other structural barriers. As a result, a moving RNA polymerase generates waves of positive supercoils ahead of the tran scription bubble and negative supercoils behind (Fig. 1c). This has been observed both in vitro and in vivo (in bacteria). In the cell, the topological problems caused by transcription are relieved through the action of topoisomerases . The two complementary DNA strands have different roles in transcription. The strand that serves as template for RNA synthesis is called the template strand. The DNA strand complementary to the template, the nontemplate strand, or coding strand, is identical in base sequence to the RNA transcribed from the gene, with U in the RNA in place of T in the DNA (Fig. 2). The coding strand for a particular gene may be located in either strand of a given chromosome (as shown in Fig. 3 for a virus). The regulatory sequences that control transcription (described later in this chapter) are by convention designated by the sequences in the coding strand.
The DNA-dependent RNA polymerase of E. coli is a large, complex enzyme with five core subunits )α2 ββ; ωMr 390,000) and a sixth subunit, one of a group designated, with variants designated by size (molecular weight). The subunit binds transiently to the core and directs the enzyme to specific binding sites on the DNA (described below). These six subunits constitute the RNA polymerase holoenzyme . The RNA polymerase holoenzyme of E. coli thus exists in several forms, depending on the type of subunit. The most common subunit is δ70 (Mr 70,000), and the upcoming discussion focuses on the corresponding RNA polymerase holoenzyme.
RNA polymerases lack a separate proofreading 3→5 exonuclease active site (such as that of many DNA polymerases), and the error rate for transcription is higher than that for chromosomal DNA replication— approximately one error for every 104 to 105 ribonucleotides incorporated into RNA. Because many copies of an RNA are generally produced from a single gene and all RNAs are eventually degraded and replaced, a mistake in an RNA molecule is of less consequence to the cell than a mistake in the permanent information stored in DNA. Many RNA polymerases, including bacterial RNA polymerase and the eukaryotic RNA polymerase II (discussed below), do pause when a mispaired base is added during transcription, and they can remove mismatched nucleotides from the 3 end of a transcript by direct reversal of the polymerase reaction. But we do not yet know whether this activity is a true proof-reading function and to what extent it may contribute to the fidelity of transcription.
FIGURE 2 Template and nontemplate (coding) DNA strands. The two complementary strands of DNA are defined by their function in transcription. The RNA transcript is synthesized on the template strand and is identical in sequence (with U in place of T) to the nontemplate strand, or coding strand.
FIGURE 3 Organization of coding information in the adenovirus genome. The genetic information of the adenovirus genome (a conveniently simple example) is encoded by a double-stranded DNA molecule of 36,000 bp, both strands of which encode proteins. The information for most proteins is encoded by the top strand—by convention, the strand transcribed from left to right—but some is en coded by the bottom strand, which is transcribed in the opposite direction. Synthesis of mRNAs in adenovirus is actually much more complex than shown here. Many of the mRNAs shown for the upper strand are initially synthesized as a single, long transcript (25,000 nucleotides), which is then extensively processed to produce the separate mRNAs. Adenovirus causes upper respiratory tract infections in some vertebrates.
