The Ribosomal Protein L2 Interacts with the RNA Polymerase Subunit and Acts as a Transcription Modulator in Escherichia coli (original) (raw)
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Journal of Bacteriology, 2010
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 protein-protein and protein-DNA interactions that often involve one or more transcription factors in addition to RNA polymerase (RNAP) subunits. The RNAP α subunit (RNAPα) 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 RNAPα protein partners should greatly increase our understanding of transcription modulation. A functional proteomics approach was employed to investigate protein components that specifically interact with RNAPα. A tagged form of Escherichia coli RNAPα 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 iden...
Transcriptional Elements Involved in the Repression of Ribosomal Protein Synthesis
Molecular and Cellular Biology, 1999
The ribosomal proteins (RPs) of Saccharomyces cerevisiae are encoded by 137 genes that are among the most transcriptionally active in the genome. These genes are coordinately regulated: a shift up in temperature leads to a rapid, but temporary, decline in RP mRNA levels. A defect in any part of the secretory pathway leads to greatly reduced ribosome synthesis, including the rapid loss of RP mRNA. Here we demonstrate that the loss of RP mRNA is due to the rapid transcriptional silencing of the RP genes, coupled to the naturally short lifetime of their transcripts. The data suggest further that a global inhibition of polymerase II transcription leads to overestimates of the stability of individual mRNAs. The transcription of most RP genes is activated by two Rap1p binding sites, 250 to 400 bp upstream from the initiation of transcription. Rap1p is both an activator and a silencer of transcription. The swapping of promoters between RPL30 and ACT1 or GAL1 demonstrated that the Rap1p binding sites of RPL30 are sufficient to silence the transcription of ACT1 in response to a defect in the secretory pathway. Sir3p and Sir4p, implicated in the Rap1p-mediated repression of silent mating type genes and of telomere-proximal genes, do not influence such silencing of RP genes. Sir2p, implicated in the silencing both of the silent mating type genes and of genes within the ribosomal DNA locus, does not influence the repression of either RP or rRNA genes. Surprisingly, the 180-bp sequence of RPL30 that lies between the Rap1p sites and the transcription initiation site is also sufficient to silence the Gal4p-driven transcription in response to a defect in the secretory pathway, by a mechanism that requires the silencing region of Rap1p. We conclude that for Rap1p to activate the transcription of an RP gene it must bind to upstream sequences; yet for Rap1p to repress the transcription of an RP gene it need not bind to the gene directly. Thus, the cell has evolved a two-pronged approach to effect the rapid extinction of RP synthesis in response to the stress imposed by a heat shock or by a failure of the secretory pathway. Calculations based on recent transcriptome data and on the half-life of the RP mRNAs suggest that in a rapidly growing cell the transcription of RP mRNAs accounts for nearly 50% of the total transcriptional events initiated by RNA polymerase II. Thus, the sudden silencing of the RP genes must have a dramatic effect on the overall transcriptional economy of the cell.
Structural basis of ribosomal RNA transcription regulation
Nature Communications, 2021
Ribosomal RNA (rRNA) is most highly expressed in rapidly growing bacteria and is drastically downregulated under stress conditions by the global transcriptional regulator DksA and the alarmone ppGpp. Here, we determined cryo-electron microscopy structures of the Escherichia coli RNA polymerase (RNAP) σ 70 holoenzyme during rRNA promoter recognition with and without DksA/ppGpp. RNAP contacts the UP element using dimerized α subunit carboxyl-terminal domains and scrunches the template DNA with the σ finger and β’ lid to select the transcription start site favorable for rapid promoter escape. Promoter binding induces conformational change of σ domain 2 that opens a gate for DNA loading and ejects σ 1.1 from the RNAP cleft to facilitate open complex formation. DksA/ppGpp binding also opens the DNA loading gate, which is not coupled to σ 1.1 ejection and impedes open complex formation. These results provide a molecular basis for the exceptionally active rRNA transcription and its vulnera...
Positive modulation of RNA polymerase III transcription by ribosomal proteins
Biochemical and Biophysical Research Communications, 2009
A yeast nuclear fraction of unknown composition, named TFIIIE, was reported previously to enhance transcription of tRNA and 5S rRNA genes in vitro. We show that TFIIIE activity co-purifies with a specific subset of ribosomal proteins (RPs) which, as revealed by chromatin immunoprecipitation analysis, generally interact with tRNA and 5S rRNA genes, but not with a Pol II-specific promoter. Only Rpl6Ap and Rpl6Bp, among the tested RPs, were found associated to a TATA-containing tRNA Ile (TAT) gene. The RPL6A gene also emerged as a strong multicopy suppressor of a conditional mutation in the basal transcription factor TFIIIC, while RPL26A and RPL14A behaved as weak suppressors. The data delineate a novel extra-ribosomal role for one or a few RPs which, by influencing 5S rRNA and tRNA synthesis, could play a key role in the coordinate regulation of the different sub-pathways required for ribosome biogenesis and functionality.
Molecular and Cellular Biology, 1989
The DNA sequences required for expression of the ribosomal protein gene rpL32 were identified by transient-expression assays of chimeric rpL32-chloramphenicol acetyltransferase genes. These studies showed that maximal rpL32 expression requires sequences in a 150- to 200-base-pair region spanning the transcriptional start site. Three discrete regions of importance were identified: one between positions -79 and -69 and two others located downstream of the transcriptional start site. Progressive 5' or 3' deletions caused stepwise decreases in expression, which suggested a complex interplay of redundant or compensatory elements. Gel mobility shift assays were used to identify trans-acting nuclear factors which bind to segments of the rpL32 promoter that are known to be important for transcription. Evidence for several distinct nuclear factors is presented. The binding sites for these factors were localized to the following regions: -79 to -69, -36 to -19, -19 to +11, +11 to +46 ...
Molecular and Cellular Biology, 1986
We have studied the protein components and nucleic acid sequences involved in stably activating the ribosomal DNA (rDNA) template and in directing accurate transcription of mammalian rRNA genes. Two protein components are necessary to catalyze rDNA transcription, and these have been extensively purified. The first, factor D, can stably associate by itself with the rDNA promoter region and is responsible for template commitment. The second component, factor C, which appears to be an activated subset of polymerase I, can stably bind to the factor D-rDNA complex but not to the rDNA in the absence of factor D. A third component which had been previously identified as a rDNA transcription factor is shown to be a RNase inhibitor. Extending our earlier observation that the approximately 150-base-pair mouse rDNA promoter consists of a minimal essential region (residues approximately -35 to approximately +9) and additional upstream stimulatory domains, we now report that each of these promot...
Nucleic Acids Research, 1989
A sequence located upstream to the E. coli rrnA P1 promoter is required for optimal promoter activity. Deletion of this sequence reduces in vivo transcription by 90%. Substitution of this upstream activating sequence with the unrelated bent DNA sequence of the kinetoplast of Crithidia fasciculata , restores in vivo expression to high levels. Cellular proteins which are present only in exponentially growing cells bind specifically to intact rmA P1, but do not bind to the promoter missing the upstream activating sequence. These proteins are associated with the 30S ribosomal subunits but can be washed off with concentrated salt. The correlation between the binding activity and cell growth rate suggests a role for these proteins in the transcriptional control of rRNA synthesis.
Molecular cell, 2016
Cell growth potential is determined by the rate of ribosome biogenesis, a complex process that requires massive and coordinated transcriptional output. In the yeast Saccharomyces cerevisiae, ribosome biogenesis is highly regulated at the transcriptional level. Although evidence for a system that coordinates ribosomal RNA (rRNA) and ribosomal protein gene (RPG) transcription has been described, the molecular mechanisms remain poorly understood. Here we show that an interaction between the RPG transcriptional activator Ifh1 and the rRNA processing factor Utp22 serves to coordinate RPG transcription with that of rRNA. We demonstrate that Ifh1 is rapidly released from RPG promoters by a Utp22-independent mechanism following growth inhibition, but that its long-term dissociation requires Utp22. We present evidence that RNA polymerase I activity inhibits the ability of Utp22 to titrate Ifh1 from RPG promoters and propose that a dynamic Ifh1-Utp22 interaction fine-tunes RPG expression to c...
Extra-Ribosomal Functions of the Ribosomal Protein, RPS3 as Predicted by In Silico Analysis
Borneo Journal of Resource Science and Technology
Products of ribosomal protein (RP) genes have been found to play extra-ribosomal roles that range from DNA repair to RNA splicing. Their association with congenital disorders or cancers has also been widely documented. However, the relatively large number of different RPs, each with perhaps unique biological roles, has compounded the comprehensive elucidation of the physiological functions of each RPs. Experimental functional studies on the many and variegated RPs are labour intensive, time-consuming and costly. Moreover, experimental studies unguided by theoretically insights entail inaccurate results. Therefore, knowledge on the actual roles of these proteins remains largely undefined. A valid alternative is the use of bioinformatics resources to computationally predict functional roles of these biomolecules. Findings from such in silico studies of the RPS3 are reported herein. We reveal an array of possible extra-ribosomal functions that includes regulation of transcription (incl...