<i>Escherichia coli</i> RNA polymerase subunit ω and its N‐terminal domain bind full‐length β′ to facilitate incorporation into the α<sub>2</sub>β subassembly

Ghosh, Parthadeb; Ishihama, Akira; Chatterji, Dipankar

doi:10.1046/j.1432-1327.2001.02381.x

Cited by 61 publications

(73 citation statements)

References 32 publications

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“…The RNAP structures indicate that there is one copy of per RNAP, and that it interacts with ␤Ј conserved regions D and G and wraps over and around the ␤Ј C-terminal tail, latching ␤Ј to the ␣ 2 ␤ subassembly (Minakhin et al 2001). The RNAP structures therefore are consistent with the model that functions as a chaperone in enzyme assembly by facilitating the binding of ␤Ј to ␣ 2 ␤ (Gentry and Burgess 1993; Mukherjee et al 1999;Ghosh et al 2001Ghosh et al , 2003. In support of this view, reconstitution of RNAP from its individual subunits is less efficient in the absence of (Mukherjee and Chatterji 1997).…”

supporting

confidence: 84%

Response of RNA polymerase to ppGpp: requirement for the ω subunit and relief of this requirement by DksA

Vrentas¹,

Gaál²,

Ross³

et al. 2005

Genes Dev.

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Previous studies have come to conflicting conclusions about the requirement for the subunit of RNA polymerase in bacterial transcription regulation. We demonstrate here that purified RNAP lacking does not respond in vitro to the effector of the stringent response, ppGpp. DksA, a transcription factor that works in concert with ppGpp to regulate rRNA expression in vivo and in vitro, fully rescues the ppGpp-unresponsiveness of RNAP lacking , likely explaining why strains lacking display a stringent response in vivo. These results demonstrate that plays a role in RNAP function (in addition to its previously reported role in RNAP assembly) and highlight the importance of inclusion of in RNAP purification protocols. Furthermore, these results suggest that either one or both of two short segments in the ␤ subunit that physically link to the ppGpp-binding region of the enzyme may play crucial roles in ppGpp and DksA function. In Escherichia coli, transcription is carried out by a multi-subunit RNA polymerase (RNAP) composed of six subunits, including two copies of ␣ and one copy each of ␤, ␤Ј, , and (for a recent review, see Geszvain and Landick 2004). ␣ 2 , ␤, ␤Ј, and comprise core RNAP, which is catalytically active but unable to recognize promoters. The ␣ 2 dimer serves as the scaffold on which ␤ and ␤Ј assemble. ␤ and ␤Ј make up the vast majority of RNAP by mass and create the enzyme's active center. To initiate transcription, one of several types of subunits, most commonly 70 , binds to core to form RNAP holoenzyme. and ␣ are site-specific DNA-binding proteins that account for specific promoter recognition. Although a high-resolution structure of E. coli RNAP has not yet been determined, X-ray structures of the Thermus aquaticus and Thermus thermophilus holoenzymes (Murakami et al. 2002b;Vassylyev et al. 2002), as well as of a T. aquaticus RNAP holoenzyme-DNA complex (Murakami et al. 2002a), elucidate how the RNAP subunits interact with each other and with template DNA., encoded by the E. coli rpoZ gene, is the smallest RNAP subunit at only 10 kDa. has homologs in all three kingdoms of life. It is present in all sequenced freeliving bacteria (although some intracellular parasitic bacteria, such as Chlamydia sp., appear to lack an homolog), in archaea (RpoK), and in eukaryotes (RPB6) (Minakhin et al. 2001). The RNAP structures indicate that there is one copy of per RNAP, and that it interacts with ␤Ј conserved regions D and G and wraps over and around the ␤Ј C-terminal tail, latching ␤Ј to the ␣ 2 ␤ subassembly (Minakhin et al. 2001). The RNAP structures therefore are consistent with the model that functions as a chaperone in enzyme assembly by facilitating the binding of ␤Ј to ␣ 2 ␤ (Gentry and Burgess 1993; Mukherjee et al. 1999;Ghosh et al. 2001Ghosh et al. , 2003. In support of this view, reconstitution of RNAP from its individual subunits is less efficient in the absence of (Mukherjee and Chatterji 1997).In contrast to the insights that the structures of RNAP provide about a role for in enzyme assembly, the str...

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supporting

confidence: 84%

Response of RNA polymerase to ppGpp: requirement for the ω subunit and relief of this requirement by DksA

Vrentas¹,

Gaál²,

Ross³

et al. 2005

Genes Dev.

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“…However, as reported with E. coli RNAP, the N-terminal domain of with 52 of 91 amino acids is capable of assembling a functional RNAP core enzyme (15). To further study the relation of rpoZ and its product with the growth of S. kasugaensis, we need to construct -null mutants.…”

Section: Discussionmentioning

confidence: 99%

The rpoZ Gene, Encoding the RNA Polymerase Omega Subunit, Is Required for Antibiotic Production and Morphological Differentiation in Streptomyces kasugaensis

et al. 2002

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The occurrence of pleiotropic mutants that are defective in both antibiotic production and aerial mycelium formation is peculiar to streptomycetes. Pleiotropic mutant KSB was isolated from wild-type Streptomyces kasugaensis A1R6, which produces kasugamycin, an antifungal aminoglycoside antibiotic. A 9.3-kb DNA fragment was cloned from the chromosomal DNA of strain A1R6 by complementary restoration of kasugamycin production and aerial hypha formation to mutant KSB. Complementation experiments with deletion plasmids and subsequent DNA analysis indicated that orf5, encoding 90 amino acids, was responsible for the restoration. A protein homology search revealed that orf5 was a homolog of rpoZ, the gene that is known to encode RNA polymerase subunit omega (), thus leading to the conclusion that orf5 was rpoZ in S. kasugaensis. The pleiotropy of mutant KSB was attributed to a 2-bp frameshift deletion in the rpoZ region of mutant KSB, which probably resulted in a truncated, incomplete of 47 amino acids. Furthermore, rpoZ-disrupted mutant R6D4 obtained from strain A1R6 by insertion of Tn5 aphII into the middle of the rpoZ-coding region produced neither kasugamycin nor aerial mycelia, similar to mutant KSB. When rpoZ of S. kasugaensis and Streptomyces coelicolor, whose deduced products differed in the sixth amino acid residue, were introduced into mutant R6D4 via a plasmid, both transformants produced kasugamycin and aerial hyphae without significant differences. This study established that rpoZ is required for kasugamycin production and aerial mycelium formation in S. kasugaensis and responsible for pleiotropy.Members of the genus Streptomyces produce an enormous variety of biologically active antibiotics and morphologically differentiate in a similar manner to eukaryotic fungi. Mature colonies of these gram-positive prokaryotic bacteria show an upper layer of aerial mycelia bearing spores that covers a lower layer of vegetative mycelia growing on the surface of agar medium. The production of antibiotics and formation of aerial hyphae, which appear to be independent events, have been considered genetically correlated based on the occurrence of pleiotropic mutants that neither produce antibiotics nor form aerial mycelia (8,17). Hence, the pleiotropic regulation has provided a subject of special interests in Streptomyces genetics.A variety of pleiotropic mutants such as bldA, bldB, and bldD mutants have been obtained from Streptomyces coelicolor together with many other types of bld mutants that fail to form aerial hyphae. Molecular cloning of the responsible genes has demonstrated transcriptional and translational regulation of pleiotropy. The bldA gene encodes leucyl tRNA for a rarely used UUA codon in streptomycetes (24, 26). Since the rare TTA codon is present in the regulatory genes for actinorhodin and undecylprodigiosin syntheses in S. coelicolor and for streptomycin and aerial mycelium productions in Streptomyces griseus, bldA mutations result in the pleiotropic phenotype in the streptomycetes (12, 23). Furthermor...

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“…has been shown to bind to regions of ␤Ј which are spread far apart over its primary sequence (13,23). The split domains remain bound to the rest of the enzyme and are able to carry out transcription, albeit at a reduced level of activity.…”

mentioning

confidence: 99%

Deletion of the Gene rpoZ , Encoding the ω Subunit of RNA Polymerase, in Mycobacterium smegmatis Results in Fragmentation of the β′ Subunit in the Enzyme Assembly

2005

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A deletion mutation in the gene rpoZ of Mycobacterium smegmatis causes reduced growth rate and a change in colony morphology. During purification of RNA polymerase from the mutant strain, the ␤ subunit undergoes fragmentation but the fragments remain associated with the enzyme and maintain it in an active state until the whole destabilized assembly breaks down in the final step of purification. Complementation of the mutant strain with an integrated copy of the wild-type rpoZ brings back the wild-type colony morphology and improves the growth rate and activity of the enzyme, and the integrity of the ␤ subunit remains unaffected.DNA-dependent RNA polymerase (RNAP) is the central enzyme involved in gene expression and also constitutes a major target for genetic regulation (7,8,31). The bacterial RNAP core enzyme consists of four subunits: alpha (␣), beta (␤), betaЈ (␤Ј) and omega () (21, 36). The subunit is the least well studied among all the subunits, though the subunit encoded by the rpoZ gene was proposed to be an integral part of the core RNAP several years ago (5, 10). In Escherichia coli is not found to be necessary for survival of the bacterium under laboratory conditions (12). At the same time homologues are present in the sequenced genomes of free-living bacteria, suggesting an important and conserved role for the protein (23).It was identified in our laboratory that is required for the restoration of denatured core RNAP to its functionally active form (25). Further, we showed that the enzyme purified from an E. coli strain lacking recruits large amounts of GroEL (26) and removal of GroEL results in a completely inactive core RNAP which lacks the ability to even associate with 70 (24). Subsequently, it was demonstrated that binds to the ␤Ј subunit and promotes RNAP assembly by facilitating the association of ␤Ј with the previous step of the assembly, ␣ 2 ␤ (13).The X-ray crystal structure of Thermus aquaticus RNAP determined at 3.3 Å resolution (36) and subsequent analysis of the -␤Ј interface by Minakhin et al. (23) identified the conserved regions of ␤Ј which binds with in a manner that reduces the configurational entropy of ␤Ј and facilitates its interaction with the ␣ 2 ␤ subassembly. Further experiments in our laboratory showed that the C-terminal tail of the subunit is constrained in the presence of ␤Ј (14).In spite of this well established evidence, a number of recent observations about the subunit in different organisms warrant suspecting functional roles for this protein which are not clearly elucidated yet. In Streptomyces kasugaensis, it was observed that a mutation in the gene encoding the subunit resulted in characteristic pleiotropic effects (19). The Kranz laboratory recently demonstrated that presence of was a prerequisite to obtain an active in vitro assembly of Rhodobacter capsulatus RNAP (30). In a completely different scenario, Periago et al. (29) demonstrated induction of YloH, the subunit of Bacillus cereus, by heat stress, suggesting a role for the subunit in stress adaptation of...

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Escherichia coli RNA polymerase subunit ω and its N‐terminal domain bind full‐length β′ to facilitate incorporation into the α₂β subassembly

Cited by 61 publications

References 32 publications

Response of RNA polymerase to ppGpp: requirement for the ω subunit and relief of this requirement by DksA

Response of RNA polymerase to ppGpp: requirement for the ω subunit and relief of this requirement by DksA

The rpoZ Gene, Encoding the RNA Polymerase Omega Subunit, Is Required for Antibiotic Production and Morphological Differentiation in Streptomyces kasugaensis

Deletion of the Gene rpoZ , Encoding the ω Subunit of RNA Polymerase, in Mycobacterium smegmatis Results in Fragmentation of the β′ Subunit in the Enzyme Assembly

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Escherichia coli RNA polymerase subunit ω and its N‐terminal domain bind full‐length β′ to facilitate incorporation into the α2β subassembly

Cited by 61 publications

References 32 publications

Response of RNA polymerase to ppGpp: requirement for the ω subunit and relief of this requirement by DksA

Response of RNA polymerase to ppGpp: requirement for the ω subunit and relief of this requirement by DksA

The rpoZ Gene, Encoding the RNA Polymerase Omega Subunit, Is Required for Antibiotic Production and Morphological Differentiation in Streptomyces kasugaensis

Deletion of the Gene rpoZ , Encoding the ω Subunit of RNA Polymerase, in Mycobacterium smegmatis Results in Fragmentation of the β′ Subunit in the Enzyme Assembly

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Escherichia coli RNA polymerase subunit ω and its N‐terminal domain bind full‐length β′ to facilitate incorporation into the α₂β subassembly