Intrinsic Ribosome Destabilization Underlies Translation and Provides an Organism with a Strategy of Environmental Sensing.

Nascent polypeptides can modulate the polypeptide elongation speed on the ribosome. Here, we show that nascent chains can even destabilize the translating Escherichia coli ribosome from within. This phenomenon, termed intrinsic ribosome destabilization (IRD), occurs in response to a special amino acid sequence of the nascent chain, without involving the release or the recycling factors. Typically, a consecutive array of acidic residues and those intermitted by alternating prolines induce IRD. The ribosomal protein bL31, which bridges the two subunits, counteracts IRD, such that only strong destabilizing sequences abort translation in living cells. We found that MgtL, the leader peptide of a Mg2+ transporter (MgtA), contains a translation-aborting sequence, which sensitizes the ribosome to a decline in Mg2+ concentration and thereby triggers the MgtA-upregulating genetic scheme. Translation proceeds at an inherent risk of ribosomal destabilization, and nascent chain-ribosome complexes can function as a Mg2+ sensor by harnessing IRD.

The Discovery of Ribosomal Protein bL31 from Escherichia coli: A Long Story Revisited.

Ribosomal protein bL31 in Escherichia coli was initially detected as a short form (62 amino acids) using Kaltschmidt and Wittmann's two-dimensional polyacrylamide gel electrophoresis (2D PAGE), but the intact form (70 amino acids) was subsequently identified by means of Wada's improved radical-free and highly reducing (RFHR) 2D PAGE, which was consistent with the analysis of its encoding gene rpmE. Ribosomes routinely prepared from the K12 wild-type strain contained both forms of bL31. ΔompT cells, which lack protease 7, only contained intact bL31, suggesting that protease 7 cleaves intact bL31 and generates short bL31 during ribosome preparation from wild-type cells. Intact bL31 was required for subunit association, and its eight cleaved C-terminal amino acids contributed to this function. 70S ribosomes protected bL31 from cleavage by protease 7, but free 50S did not. In vitro translation was assayed using three systems. The translational activities of wild-type and ΔrpmE ribosomes were 20% and 40% lower than those of ΔompT ribosomes, which contained one copy of intact bL31. The deletion of bL31 reduces cell growth. A structural analysis predicted that bL31 spans the 30S and 50S subunits, consistent with its functions in 70S association and translation. It is important to re-analyze in vitro translation with ribosomes containing only intact bL31.

Autogenous regulation in vivo of the rpmE gene encoding ribosomal protein L31 (bL31), a key component of the protein-protein intersubunit bridge B1b.

Bacterial ribosomal proteins (r-proteins) encoded by nonessential genes often carry out very important tasks in translation. In particular, this is the case of a small basic bacteria-specific r-protein L31 (bL31). Recent studies revealed a crucial role of bL31 in formation of the protein-protein intersubunit bridge B1b and hence its contribution to ribosome dynamics. Our goal was to study in vivo regulation of the rpmE operon encoding bL31. We used a previously developed approach based on chromosomally integrated fusions with the lacZ reporter. E. coli rpmE is transcribed from two promoter regions, and translation of both mRNA transcripts was shown to be feedback regulated by bL31, indicating that the autogenous operator is located within the shorter transcript. The bL31-mediated control of rpmE is gene-specific, as no regulation was found for rpmE-unrelated reporters. Thus, bL31, as many other r-proteins, possesses dual activity in living cells, acting both as an integral ribosome component and an autogenous repressor. Phylogenetic studies revealed the presence of a highly conserved stem-loop structure in the rpmE 5'UTR, a presumable translational operator targeted by bL31, which was further confirmed by site-directed mutagenesis. This stable operator stem-loop separates an AU-rich translational enhancer from a Shine-Dalgarno element, which is a rare case of a noncontiguous translation initiation region. Sequence/structure computational approaches classify bL31 as an RNA-binding protein, consistent with its repressor function discovered here. Mutational analysis of bL31 showed that its unstructured amino-terminal part enriched in lysine is necessary for the repressor activity.

Promotion on labor process and relief of the low back pain by relaxing pelvic muscle with Shangliao (BL 31) point injection in women using epidural analgesia during labor: A randomized, controlled, clinical trial.

BACKGROUND: The purpose of this study was to explore the effects of combing Shangliao point injection with epidural analgesia on labor pain and birth process in women with low back pain and the possible mechanisms. METHODS: 93 consecutive women were randomized to receive either Shangliao point injection combined with epidural analgesia or epidural analgesia. Another 14 women were recruited to explore the mechanisms and the transperineal ultrasound was performed accordingly. RESULTS: The main result duration from epidural analgesia to baby delivery was significantly shorter in epidural analgesia and saline injection group than that in epidural analgesia group 307.0 (175.0-445.0) min VS 369.0 (254.0-563.0) min (P = 0.02). The verbal numerical rate scaling score in low back during the first contraction was significantly decreased 5.0 (4.0-7.0) after Shangliao point injections (P < 0.001). The consumption of ropivacaine per hour was significantly less in epidural analgesia and saline injection group than in epidural analgesia group (-0.4 mg, 95%CI: -0.1 to -1.8; P = 0.03). The angle of progression and anteroposterior diameter of the levator hiatus at rest and during valsalva were significantly increased after shangliao point injection (7.10°, 95%CI, 1.50~12.70; P = 0.02); (9.10°, 95%CI, 3.60~14.58; P < 0.01); (0.27 cm, 95%CI, 0.03~0.51; P = 0.03); (0.30 cm, 95%CI, 0.13~0.48; P < 0.01). CONCLUSIONS: Shangliao point injection could shorten the time to baby delivery and rapidly relieve low back pain in addition to epidural analgesia, that may attribute to its function of relaxing the pelvic floor muscles and promote fetal head progress.

Bacterial ribosome heterogeneity: Changes in ribosomal protein composition during transition into stationary growth phase.

Ribosomes consist of many small proteins and few large RNA molecules. Both components are necessary for ribosome functioning during translation. According to widely accepted view, bacterial ribosomes contain always the same complement of ribosomal proteins. Comparative bacterial genomics data indicates that several ribosomal proteins are encoded by multiple paralogous genes suggesting structural heterogeneity of ribosomes. In E. coli, two r-proteins bL31 and bL36 are encoded by two genes: rpmE and ykgM encode bL31 protein paralogs bL31A and bL31B, and rpmJ and ykgO encode bL36 protein paralogs bL36A and bL36B respectively. We have found several similarities and differences between ribosomes of exponential and stationary growth phases by using quantitative mass spectrometry and X-ray crystallography. First, composition of ribosome associating proteins changes profoundly as cells transition from exponential to stationary growth phase. Ribosomal core proteins bL31A and bL36A are replaced by bL31B and bL36B, respectively. Second, our X-ray structure of the 70S ribosome demonstrates that bL31B and bL36B proteins have similar ribosome binding sites to their A counterparts. Third, ribosome subpopulations containing A or B paralogs existed simultaneously demonstrating that E. coli ribosomes are heterogeneous with respect to their paralogous ribosomal protein composition that changes via protein exchange.

The Intersubunit Bridge B1b of the Bacterial Ribosome Facilitates Initiation of Protein Synthesis and Maintenance of Translational Fidelity.

In bacteria, ribosomal subunits are connected via 12 intersubunit bridges involving RNA-RNA, RNA-protein, and protein-protein interactions. The only protein-protein bridge in the ribosome is ribosomal intersubunit bridge 1b (B1b), which is mainly formed by the bacterial protein L31 (bL31) and connects the head domain of 30S subunit and the central protuberance of the 50S subunit. It is known to be the most dynamic intersubunit bridge. Here, we have evaluated the role of bL31 and thereby the bridge B1b in the working cycle of the ribosome. First, bL31-deficient ribosomes are severely compromised in their ability to ensure translational fidelity particularly in reading frame maintenance in vivo. Second, in the absence of bL31, the rate of initiation is significantly reduced both in vivo and in vitro. Third, polysome profile and subunit reassociation assays demonstrate that bL31 is important for stabilizing subunit joining in vivo and in vitro. Together, our results demonstrate that bL31 is important for determining translational fidelity and stabilizing subunit association. We conclude that the only protein-protein intersubunit bridge of the bacterial ribosome facilitates translation initiation and is essential for maintaining the reading frame of mRNA translation.