Efficient assembly of ribosomes is inhibited by deletion of bipA in Escherichia coli.

UNLABELLED: The bacterial BipA protein belongs to the EF-G family of translational GTPases and has been postulated to be either a regulatory translation factor or a ribosome assembly factor. To distinguish between these hypotheses, we analyzed the effect of bipA deletion on three phenotypes associated with ribosome assembly factors: cold sensitivity, ribosome subunit distribution, and rRNA processing. We demonstrated that a ΔbipA strain exhibits a cold-sensitive phenotype that is similar to, and synergistic with, that of a strain with a known ribosome assembly factor, deaD. Additionally, the bipA deletion strain displayed a perturbed ribosome subunit distribution when grown at low temperature, similar to that of a deaD mutant, and again, the double mutant showed additive effects. The primary ribosomal deficiency noted was a decreased level of the 50S subunit and the appearance of a presumed pre-50S particle. Finally, deletion of bipA resulted in accumulation of pre23S rRNA, as did deletion of deaD. We further found that deletion of rluC, which encodes a pseudouridine synthase that modifies the 23S rRNA at three sites, suppressed all three phenotypes of the bipA mutant, supporting and extending previous findings. Together, these results suggest that BipA is important for the correct and efficient assembly of the 50S subunit of the ribosome at low temperature but when unmodified by RluC, the ribosomes become BipA independent for assembly. IMPORTANCE: The ribosome is the complex ribonucleoprotein machine responsible for protein synthesis in all cells. Although much has been learned about the structure and function of the ribosome, we do not fully understand how it is assembled or the accessory proteins that increase efficiency of biogenesis and function. This study examined one such protein, BipA. Our results indicate that BipA either directly or indirectly enhances the formation of the 50S subunit of the ribosome, particularly at low temperature. In addition, ribosomes contain a large number of modified nucleosides, including pseudouridines. This work demonstrates that the function of BipA is tied to the modification status of the ribosome and may help us understand why these modifications have been retained.

Suppression of DeltabipA phenotypes in Escherichia coli by abolishment of pseudouridylation at specific sites on the 23S rRNA.

The BipA protein of Escherichia coli has intriguing similarities to the elongation factor subfamily of GTPases, including EF-Tu, EF-G, and LepA. In addition, phenotypes of a bipA deletion mutant suggest that BipA is involved in regulation of a variety of pathways. These two points have led to speculation that BipA may be a novel regulatory protein that affects efficient translation of target genes through direct interaction with the ribosome. We isolated and characterized suppressors of the cold-sensitive growth phenotype exhibited by DeltabipA strains and identified insertion mutations in rluC. The rluC gene encodes a pseudouridine synthase responsible for pseudouridine modification of 23S rRNA at three sites, all located near the peptidyl transferase center. Deletion of rluC not only suppressed cold sensitivity but also alleviated the decrease in capsule synthesis exhibited by bipA mutants, suggesting that the phenotypic effects of BipA are manifested through an effect on the ribosome. The suppressor effect is specific to rluC, as deletion of other rlu genes did not relieve cold sensitivity, and further, more than a single pseudouridine residue is involved, as alteration of single residues did not produce suppressors. These results are consistent with a role for BipA in either the structure or the function of the ribosome and imply that wild-type ribosomes are dependent on BipA for efficient expression of target mRNAs and that the lack of pseudouridylation at these three sites renders the ribosomes BipA independent.

The SecY translocation complex: convergence of genetics and structure.

All organisms share a requirement for translocation of proteins across membranes. The major mechanism for this process is the universally conserved SecY/Sec61 pathway. Many years of extensive genetic and biochemical analyses identified the components of the SecY/Sec61 pathway, demonstrated that most exported proteins use this route for translocation, and led to understanding of many functions of the components. Recently, structural predictions based on genetic analyses in Escherichia coli were confirmed, in a striking and satisfying manner, by the solution of an X-ray crystal structure from an archaeal SecY complex. This review discusses the genetic background that led to those hypotheses and the convergence of genetic studies with structural data.

Modeling the effects of prl mutations on the Escherichia coli SecY complex.

The apparatus responsible for translocation of proteins across bacterial membranes is the conserved SecY complex, consisting of SecY, SecE, and SecG. Prior genetic analysis provided insight into the mechanisms of protein export, as well as the interactions between the component proteins. In particular, the prl suppressor alleles of secE and secY, which allow export of secretory proteins with defective signal sequences, have proven particularly useful. Here, we report the isolation of novel mutations in secE and secY, as well as the phenotypic effects of combinations of prl mutations. These new alleles, as well as previously characterized prl mutations, were analyzed in light of the recently published crystal structure of the archaeal SecY complex. Our results support and expand a model of Prl suppressor activity that proposes that all of the prlA and prlG alleles either destabilize the closed state of the channel or stabilize the open form. These mutants thus allow channel opening to occur without the triggering event of signal sequence binding that is required in a wild-type complex.

Decreased gene expression from T7 promoters may be due to impaired production of active T7 RNA polymerase.

BACKGROUND: Protein expression vectors that utilize the bacteriophage T7 polymerase/promoter system are capable of very high levels of protein production. Frequently, however, expression from these vectors does not reliably achieve optimal levels of protein production. Strategies have been proposed previously that successfully maintain high expression levels, however we sought to determine the cause of induction failure. RESULTS: We demonstrated that decreases in protein overproduction levels are not due to significant plasmid loss nor to mutations arising on the plasmid, but instead largely are attributable to chromosomal mutations that diminish the level of functional T7 RNA polymerase, resulting in decreased expression from the plasmid. Isolation of plasmid DNA from non-expressing strains and reintroduction of the plasmid into a T7 RNA polymerase-producing strain such as BL21(lambdaDE3) reproducibly restored high level protein production. CONCLUSIONS: Our results suggest that a major contributing factor to decreased expression levels in T7 based systems is chromosomal mutation resulting in loss of functional T7 RNA polymerase. Consistent with this hypothesis, we found that optimal protein overproduction was obtained reproducibly from T7 promoters using freshly transformed cells that had not been subjected to outgrowth during which mutations could accumulate.

Recognition of secretory proteins in Escherichia coli requires signals in addition to the signal sequence and slow folding.

BACKGROUND: The Sec-dependent protein export apparatus of Escherichia coli is very efficient at correctly identifying proteins to be exported from the cytoplasm. Even bacterial strains that carry prl mutations, which allow export of signal sequence-defective precursors, accurately differentiate between cytoplasmic and mutant secretory proteins. It was proposed previously that the basis for this precise discrimination is the slow folding rate of secretory proteins, resulting in binding by the secretory chaperone, SecB, and subsequent targeting to translocase. Based on this proposal, we hypothesized that a cytoplasmic protein containing a mutation that slows its rate of folding would be recognized by SecB and therefore targeted to the Sec pathway. In a Prl suppressor strain the mutant protein would be exported to the periplasm due to loss of ability to reject non-secretory proteins from the pathway. RESULTS: In the current work, we tested this hypothesis using a mutant form of lambda repressor that folds slowly. No export of the mutant protein was observed, even in a prl strain. We then examined binding of the mutant lambda repressor to SecB. We did not observe interaction by either of two assays, indicating that slow folding is not sufficient for SecB binding and targeting to translocase. CONCLUSIONS: These results strongly suggest that to be targeted to the export pathway, secretory proteins contain signals in addition to the canonical signal sequence and the rate of folding.