Mutants in ABC10β, a Conserved Subunit Shared by All Three Yeast RNA Polymerases, Specifically Affect RNA Polymerase I Assembly

Abstract

ABC10␤, a small polypeptide common to the three yeast RNA polymerases, has close homology to the N subunit of the archaeal enzyme and is remotely related to the smallest subunit of vaccinial RNA polymerase. The eucaryotic, archaeal, and viral polypeptides share an invariant motif CX 2 C. . . CC that is strictly essential for yeast growth, as shown by site-directed mutagenesis, whereas the rest of the ABC10␤ sequence is fairly tolerant to amino acid replacements. ABC10␤ has Zn 2؉ binding properties in vitro, and the CX 2 C . . . CC motif may therefore define an atypical metal-chelating site. Hybrid subunits that derive most of their amino acids from the archaeal subunit are functional in yeast, indicating that the archaeal and eucaryotic polypeptides have a largely equivalent role in the organization of their respective transcription complexes. However, all eucaryotic forms of ABC10␤ harbor a HVDLIEK motif that, when mutated or replaced by its archaeal counterpart, leads to a polymerase I-specific lethal defect in vivo. This is accompanied by a specific lack in the largest subunit of RNA polymerase I (A190) in cell-free extracts, showing that the mutant enzyme is not properly assembled in vivo.The nuclear genome of eucaryotes is transcribed by three heteromultimeric RNA polymerases that respectively contain 14, 12, and 17 distinct subunits in Saccharomyces cerevisiae. The two largest subunits are related to the ␤Ј and ␤ components of the ␣ 2 ␤␤Ј bacterial core enzyme and to the equivalent subunits of the archaeal and vaccinial RNA polymerases. Biochemical and genetic studies have established that they harbor the active site of the yeast (1, 2) and bacterial (3) enzymes. Homology to the bacterial ␣ subunit, although less pregnant, was also observed with the eucaryotic and archaeal enzymes (4, 5). The ␣ 2 ␤␤Ј core polymerase structure is therefore preserved in all eucaryotic, archaeal, bacterial, and viral forms of RNA polymerases. A number of additional subunits are structurally conserved or even strictly identical from one to another polymerases (6 -8) and are functionally conserved from yeast to man (9, 10). Most of them are structurally related to known components of the archaeal polymerase, indicating the existence of an extended core enzyme form that is common to the archaeal and eucaryotic lineages (5).The present work deals with ABC10␤, a small polypeptide of 70 amino acids that is shared by all three yeast RNA polymerases, is able to bind Zn 2ϩ in vitro (11) and is essential for growth (12). We have previously determined the amino acid sequence of that polypeptide (13), which has a close homology to the N subunit of the archaeal enzyme (5,10,14) and is remotely related to the smallest subunit of vaccinial RNA polymerase (15). We report here that the yeast subunit and its archaeal homolog are largely interchangeable in vivo and that the eucaryotic, archaeal, and viral polypeptides have in common an invariant metal binding domain (CX 2 C . . . CC) critical for its biological activity in S. cerevisiae. Moreov...

Subunits Common to RNA Polymerases

“…Rpb10 has a strong conservation along eukaryotic sequences with 41 identical amino acid positions in fungal, plant and human sequences [73]. In addition, Rpb10 shows a close homology to the N subunit of archaeal enzyme [12,54,74] and is loosely related to the smallest enzyme of cytoplasmic DNA viruses [73,75,76].…”

“…In vivo studies in budding yeast have demonstrated that Rpb10 can be functionally replaced by its human homologue (RPB10) [12]. Nevertheless, the N subunit of archaeal cannot replace Rpb10 in vivo [73]. However, yeast/archaeal chimeras are largely interchangeable, pointing to a conserved function in their respective transcription complexes [12].…”

“…The Rpb10 sequence also harbours an atypical and invariant metal-binding domain CX 2 C…CC with Zn 2+ binding properties in vitro [71,73] that is conserved in eukaryotic, archaeal and viral polypeptides and that is strictly essential for yeast growth, as shown in site-directed mutagenesis experiments [73]. Curiously, mutations out of the metal-chelating domain sequence are fairly tolerant to amino acid replacements [73].…”

“…Rpb10 and Rpb12 fill concave depressions of Rpb2 and thereby act as structural adaptors between Rpb2 and Rpb3 (reviewed in [1]). Notably, mutations of the invariant HVDLIEK motif lead to a complete depletion of the largest RNA pol I subunit (Rpa190) and decrease the accumulation of mature rRNA species transcribed by RNA pol I [73]. However, Rpb10 could have additional functions beyond RNA polymerases assembly.…”

“…All the eukaryotic forms of Rpb10 share an invariant HVDLIEK motif (located between positions His-53 and Pro-65 in S. cerevisiae) critical for the biological activity of Rpb10 [73]. The Rpb10 sequence also harbours an atypical and invariant metal-binding domain CX 2 C…CC with Zn 2+ binding properties in vitro [71,73] that is conserved in eukaryotic, archaeal and viral polypeptides and that is strictly essential for yeast growth, as shown in site-directed mutagenesis experiments [73].…”