Читать книгу Snyder and Champness Molecular Genetics of Bacteria - Tina M. Henkin - Страница 142

The transcription of DNA into RNA is carried out by RNA polymerase. In bacteria, the same RNA polymerase makes all the cellular RNAs, including rRNA, tRNA, and mRNA (but excluding the RNA primers used in DNA replication). There are approximately 2,000 molecules of RNA polymerase in each bacterial cell under slow growth conditions, with higher numbers during rapid growth. Only the primer RNAs of Okazaki fragments are made by a different RNA polymerase (primase) during DNA replication. In contrast, eukaryotes have three nuclear RNA polymerases, as well as a mitochondrial RNA polymerase, that make their cellular RNAs.

Figure 2.3 shows a schematic structure of Escherichia coli RNA polymerase, which has six subunits and a molecular mass of more than 400,000 Daltons (Da) (400 kDa), making it one of the largest enzymes in the E. coli cell. The core enzyme consists of five subunits: two identical α subunits, two very large subunits called β and β′, and the ω subunit. The α, β, and β′ subunits are essential parts of the RNA polymerase, and the ω subunit helps in its assembly. A sixth subunit, the σ factor, is required only for initiation and cycles off the enzyme after initiation of transcription. Without the σ factor, the RNA polymerase is called the core enzyme; with σ, it is called the holoenzyme.

Crystal structures of RNA polymerase from Thermus aquaticus and E. coli have revealed the structure shown in Figure 2.4. The overall shape resembles a crab claw. Regions of the β and β′ subunits form the pincers of the crab claw (as shown in Figure 2.3). The two α subunits, αI and αII, are on the opposite end from the claw, making up the rear end when the enzyme is transcribing DNA. The carboxy-terminal regions of the α polypeptides hang out from the enzyme, where they can contact other proteins or DNA regions upstream of the promoter and stimulate transcription initiation (see below). The ω subunit is also on this end of the RNA polymerase, wrapped around the β′ subunit (Figure 2.3).

The σ factor is crucial for directing RNA polymerase to recognize the promoter sequence, which is the site on the DNA at which transcription initiates. When the σ factor is bound to the complex to form the holoenzyme, it wraps around the front end of the core enzyme in such a way that it can contact the DNA as it enters the open claw. One of the σ domains, domain σ₂, contacts the β′ pincer and is in position to bind to one part of the promoter sequence on the DNA (the –10 region), while two other domains, σ₃ and σ₄, contact the β subunit further upstream in the active-center channel in such a way that the σ₄ domain is in position to contact the –35 region of the promoter (see below). The RNA polymerases of all bacteria are probably very similar in sequence and composition. One interesting difference is that in some types of bacteria, the β and β′ subunits are attached to each other to form an even larger polypeptide. The eukaryotic and archaeal core RNA polymerases have more subunits and have very different sequences, but their basic overall structures are very similar to that of bacterial RNA polymerase.

Figure 2.3 The structure of bacterial RNA polymerase. The core enzyme is composed of the β, β′, ω, and two copies of the α protein. Addition of the σ protein results in formation of the holoenzyme, which is the form required for initiation of transcription. The functions of some of the domains of σ, σ₁, and σ_3.2 are discussed in this chapter.

Figure 2.4 Crystal structure of bacterial RNA polymerase and σ interactions with promoter DNA. Subunits of RNA polymerase are shown. The α subunits are divided into the α subunit amino-terminal domains (αNTD), which associate with the β and β′ subunits, and the α subunit carboxy-terminal domains (αCTD), which can bind to the UP element in the DNA. The structures of the αCTDs are not resolved and are shown as balls. σ is divided into four domains that extend along the promoter DNA, interacting with different regions of the promoter. From Murakami KS, Masuda S, Campbell EA, et al, Science 296:1285–1290, 2002. Modified with permission from AAAS.