Structural Basis of Transcription: RNA Polymerase II at 2.8 Ångstrom Resolution

Abstract

Structures of a 10-subunit yeast RNA polymerase II have been derived from two crystal forms at 2.8 and 3.1 angstrom resolution. Comparison of the structures reveals a division of the polymerase into four mobile modules, including a clamp, shown previously to swing over the active center. In the 2.8 angstrom structure, the clamp is in an open state, allowing entry of straight promoter DNA for the initiation of transcription. Three loops extending from the clamp may play roles in RNA unwinding and DNA rewinding during transcription. A 2.8 angstrom difference Fourier map reveals two metal ions at the active site, one persistently bound and the other possibly exchangeable during RNA synthesis. The results also provide evidence for RNA exit in the vicinity of the carboxyl-terminal repeat domain, coupling synthesis to RNA processing by enzymes bound to this domain.

RNA polymerase I activation and hibernation: unique mechanisms for unique genes

“…Below the active site, located at the centre of the enzyme, a secondary pore opens in the floor of the cleft. In Pol II, the pore was suggested to constitute the entry site for NTPs [26] and shown to harbour extruded RNA during backtracking [27]. Next to the pore, the bridge helix crosses the cleft in the vicinity of the trigger loop.…”

“…On the left, the 14 subunits forming the complex are coloured according to the top legend. The middle panel shows the 4 modules defined for Pol II [26] as well as the stalk and the A49/A34.5 dimerization region. On the right, the most important structural domains are labelled.…”

“…In analogy to Pol II and bacterial RNA polymerase [26,28], four mobile modules can be defined in Pol I (Figure 1(A)). The core module harbours essential elements for RNA polymerization such as the active centre and the pore, as well as several subunits and regions involved in enzyme assembly, including subunits AC40, AC19, Rpb10 and Rpb12.…”

“…The clamp essentially comprises domains of A190 with a minor contribution of A135. In contrast to Pol II where clamp swinging over the shelf was shown to play a role in transcriptional regulation [26], the clamp and shelf modules in Pol I constitute a rigid mobile unit [22]. Because the Pol I stalk, formed by the A43/A14 heterodimer, is tightly attached to both the shelf and the clamp, these two modules and the stalk can be regarded as a single unit that pivots against the core and jaw-lobe modules for cleft closure [20,25].…”

Protein factors in pre-mRNA 3′-end processing

“…The repeated serine residues within the CTD are susceptible to phosphorylation [207]. The CTD is not observed in the structure of yeast PolII, suggesting that it may be flexible [208]. Sequence analysis and early NMR studies proposed that the CTD forms a β-turn.…”