Following protein synthesis inhibition in cycloheximide growth-arrested yeast cells, the rates of tRNA and 5 S RNA synthesis decrease with apparent half-times of about 20 and 10 min, respectively. This effect is mimicked by extracts of treated cells, and the impairment of tRNA gene transcription activity that is observed in vitro parallels the in vivo inactivation of RNA polymerase III transcription. As revealed by experiments in which partially purified class III transcription factors were singly added to extracts of treated cells, only the activity of the multiprotein transcription factor TFIIIB is severely impaired after 3 h of cycloheximide treatment. Similar assays carried out in an in vitro transcription system in which TFIIIB activity was reconstituted by a combination of the TATA box-binding protein (TBP), the 70-kDa component TFIIIB70, plus a partially purified fraction known as B" have shown that the latter two components are both necessary and sufficient to restore control levels of transcription. Their activity, but not TBP activity, is considerably reduced in extracts of treated cells. TFIIIB70 and a component of fraction B" thus appear to be the selective targets of the down-regulation of polymerase III transcription that is brought about by cycloheximide. A substantial depletion of the TFIIIB70 polypeptide was detected by Western immunoblot analysis of extracts derived from cycloheximide growth-arrested cells, indicating that the inactivation of this TFIIIB component results primarily from its enhanced destabilization under conditions of protein synthesis inhibition.

SummaryBiochemical and crystallographic data suggest that, in contrast with other organisms, the active maize protein kinase CK2 might be composed simply of a catalytic polypeptide (CK2a), thus lacking CK2b regulatory subunits. To investigate the existence and functionality of CK2b regulatory subunits in Zea mays, we have screened a maize cDNA library using different approaches and have isolated three fulllength cDNAs encoding CK2b regulatory subunits (CK2b-1, CK2b-2 and CK2b-3) and a cDNA coding for a novel CK2a catalytic subunit, CK2a-3. The pattern of expression of all these a/b subunits has been studied in different organs and developmental stages using speci®c probes for each isoform, and indicates that while CK2a subunits are constitutive, CK2b subunits are expressed differentially during embryo development. The yeast two-hybrid system and pull-down assays have been used to study speci®c interactions between the different subunits. While CK2a subunits are unable to self-associate, preferential interactions between a/b isoforms and b/b isoforms can be predicted. Furthermore, we show that maize CK2a/b subunits assemble into a structural tetrameric complex which has very similar properties to those described in other organisms, and that expression of maize CK2b subunits in yeast allows the rescue of the phenotypic defects associated to the lack of CK2 function, thus demonstrating the functionality of maize CK2b regulatory subunits.

No abstract

In plants, protein kinase CK2 is involved in different processes that control many aspects of metabolism and development. In mammals and yeast the enzyme is a heterotetramer composed of two types of subunits. During years the subunit composition of the maize protein kinase CK2 enzyme has been a source of controversy. We have recently characterized the maize holoenzyme subunits. Our results show that multiple catalytic and regulatory subunits are expressed in maize and are able to specifically interact with other alpha and beta subunits suggesting a high level of heterogeneity in the typical heterotetrameric structure. Here, we summarize data available on plant CK2 enzymes, in order to clarify the distinctive features and functions of plant protein kinase CK2.