The Ribosomal Protein L2 Interacts with the RNA Polymerase α Subunit and Acts as a Transcription Modulator in Escherichia coli

Abstract

Identification of interacting proteins in stable complexes is essential to understand the mechanisms that regulate cellular processes at the molecular level. Transcription initiation in prokaryotes requires coordinated proteinprotein and protein-DNA interactions that often involve one or more transcription factors in addition to RNA polymerase (RNAP) subunits. The RNAP ␣ subunit (RNAP␣) is a key regulatory element in gene transcription and functions through direct interaction with other proteins to control all stages of this process. A clear description of the RNAP␣ protein partners should greatly increase our understanding of transcription modulation. A functional proteomics approach was employed to investigate protein components that specifically interact with RNAP␣. A tagged form of Escherichia coli RNAP␣ was used as bait to determine the molecular partners of this subunit in a whole-cell extract. Among other interacting proteins, 50S ribosomal protein L2 (RPL2) was clearly identified by mass spectrometry. The direct interaction between RNAP␣ and RPL2 was confirmed both in vivo and in vitro by performing coimmunoprecipitation and bacterial two-hybrid experiments. The functional role of this interaction was also investigated in the presence of a ribosomal promoter by using a ␤-galactosidase gene reporter assay. The results clearly demonstrated that RPL2 was able to increase ␤-galactosidase expression only in the presence of a specific ribosomal promoter, whereas it was inactive when it was assayed with an unrelated promoter. Interestingly, other ribosomal proteins (L1, L3, L20, and L27) did not have any effect on rRNA expression. The findings reported here strongly suggest that in addition to its role in ribosome assembly the highly conserved RPL2 protein plays a specific and direct role in regulation of transcription.Understanding the mechanism and regulation of transcription in bacteria requires dissection of the specific roles of the individual components of the multiprotein transcription complex, including the multisubunit enzyme RNA polymerase (RNAP). It is well known, in fact, that one of the major factors ensuring correct gene expression in microorganisms is the efficiency with which RNAP recognizes the specific promoters of different genes.The single form of RNAP in Escherichia coli consists of a tetrameric core enzyme (␤␤Ј␣2) that is capable of RNA synthesis and factor-independent termination and a subunit that is responsible for recognition of specific transcription initiation sites. However, although the RNAP subunit is the main determinant for promoter recognition, the ␣ subunit also plays a key role in the stability of the transcription complex (3, 10).The E. coli RNAP ␣ subunit (RNAP␣) consists of two domains, an amino-terminal domain (NTD) and a carboxy-terminal domain (CTD), that are connected by a flexible linker that allows the CTD to interact with promoter elements located different distances from the RNAP binding site (2). The ␣ subunit has at least two functions during gene transcription; ...

Transcription–translation coupling: direct interactions of RNA polymerase with ribosomes and ribosomal subunits

“…The interface between the polymerase and the large subunit is located on the face that binds the small subunit, which, upon association with the small subunit, is buried within the ribosome (Figure 4B and C ). Modeling the binding of RNA polymerase on the large ribosomal subunit places the RNA polymerase in close proximity to the ribosomal protein L2, which is known to bind the α subunit of RNA polymerase ( 72 ). The set of proteins crosslinked in the complex with ribosomes overlaps with that of the small ribosomal subunit complex (Figure 3C and D ), clustering around the mRNA exit site of the ribosome (Figure 4A and C ), hinting at the possible coordination between the transcription and translation initiation of the nascent RNA.…”

Enhanced expression of Bacillus subtilis yaaA can restore both the growth and the sporulation defects caused by mutation of rplB, encoding ribosomal protein L2

“…(c) Crude cell extracts were sedimented through a 10-40 % sucrose gradient as described in the Supplementary Methods. replication by inhibiting oligomer formation of DnaA (Rippa et al, 2010;Chodavarapu et al, 2011). Taken together, the experimental data indicate that ribosomal protein L2 is a multifunctional protein.…”

Protein Interactomes Identify Distinct Pathways forStreptococcus mutansYidC1 and YidC2 Membrane Protein Insertases

“…In the current study, the large ribosomal subunit protein, L2, was the most abundant ribosomal protein pulled down by both the GST-YidC1CT and GST-YidC2CT fusion polypeptides. In E. coli , L2 not only acts as a structural component of the ribosome, it is also processed to a truncated derivative (tL2) that can interact with the RNA polymerase alpha subunit and modulate transcription [52]. E. coli L2 has also been reported to interact with the Hsp90 homolog HtpG to modulate its ATPase activity, and also to bind to other chaperones including DnaK/DnaJ/GrpE and GroEL/GroES [53].…”