Sunday, August 11, 2013

NusA to save the day during heat shock: commentary and review of Li, et al 2013

A review of Li, et al Escherichia coli transcription termination factor NusA: heat-induced oligomerization and chaperone activity (2013) Scientific Reports, vol 3 (2347) PMCID: PMC3731644

In previous blog posts, I have taken a look at two slightly dated articles concerning drug development against mycobacterium tuberculosis. Here I will continue my series of reviews but with a new direction. My last review provided a new perspective on a study that had already received press from others. For this review, I have chosen a much more recent paper, which has not been analyzed (to my knowledge) by another other blogger, science journalist, or microbiology enthusiast.

Why have I chosen a paper on NusA molecular biology in E. coli? Other than the fact that the article is fresh off the press (from the Nature sub-journal Scientific Reports), the claims (which are fairly well supported) the authors make are another example of how bacterial proteins often function as swiss army knifes: they have multiple functions, sometimes not revealed until environmental conditions are changed or cellular stresses are introduced. 

I will begin this review with a short summary, known as a Capsule. This style of synopsis / abstract is being pioneered by the Journal for Biological Chemistry (JBC), and I think it is a great idea for making summaries primary research literature more accessible to a general audience. One way to think of it is a shorter abstract, written not for experts but for the public (and policy makers, I suppose!). Authors of manuscripts submitted to JBC must provide a capsule statement; here, the capsule below is my own, not written by the authors of the NusA study in Scientific Reports (and not conforming to JBC's strict 60 word limit).


Background:  NusA is a protein factor known to be involved in transcription termination and anti-termination (transcription is part of the process of turning genetic information in DNA into proteins and enzymes).

Results: Upon heat shock, NusA forms oligomers (multiple copies of the same protein factor bound together) which help prevent other proteins from aggregating.  

Conclusion: NusA contributes to the heat-shock resistance in E. coli by acting as a buffer to protein aggregation.

Significance: Describes a new role for NusA and expands the knowledge of how bacteria cope with stress; these abilities (in general) are important for many bacteria, including pathogenic bacteria that must resist stress from our immune system and medicines.

I invite readers to form their own capsule of this article, especially if you disagree with my choice of areas to emphasis.

Manuscript Highlights

1. NusA is the latest example of a multi-functional protein with latent chaperone 'buffer' activity during heat-shock. GreA, another transcription related factor, is also recent example.

2. NusA oligomerization (distinct from aggregation), mediated by the C-terminal repeat domains, is thought to be responsible for the chaperone buffer activity.

3. NusA's role in heat-shock conditions is not demonstrated under physiological conditions; experiments are done in vitro or with over-expressed and tagged NusA. This may reflect technical limitations.

Select 'Read More' to see the rest of the review


In this study from the Laboratory groups of Dr. Ping Xu and Dr. Yanhe Ma at Chinese Universities in Beijing and Shanghai, respectively, the heat shock oligomerization and chaperone activity of NusA is examined. This work continues to elucidate the way in which bacteria deal with heat stress, which has the ability to cause protein aggregation. Presumably, other aggregation promoting stresses can also be mitigated in similar manners. 

NusA is a protein factor known to play important roles in transcription termination (and anti-termination, depending on the genetic context!). The authors of this study follow at least one lead from their previous work in which another transcription factor, GreA, was shown to have an alternative, heat-protective function (Li, 2012). Indeed, GreA was known to be involved in bacterial stress responses (including acid, salt and cold stress.) The idea that a transcription factor such as GreA could act as a chaperone is suggested by the characterization of sHSP (small heat-shock proteins) that are found among diverse functional groups of E coli proteins (including translation factors such as EF-G, EF-Tu, Thioredoxin and the ribosome itself; Li, 2010; Kern, 2003).

In this study, the authors purify NusA and examine the size and organization of NusA complexes after subjecting the purified protein to heat shock conditions. Instead of aggregating into massive tangles that would be disruptive to cellular functions, NusA appears to oligomerize in vitro into soluble complexes of 10-12 monomers. These NusA oligomers (which are stable at room temperature once formed) appear to have a chaperone buffer activity, in that they are able to prevent the aggregation of other cellular proteins. 

How many functions does NusA have?

This work claims that NusA has a heat-shock chaperone function, in addition to the known transcription related roles of this protein. This is an interesting idea, not unique to NusA. Many bacterial proteins have been shown to have multi-functional roles, which is plausible considering their small proteomes compared to some eukaryotic counterparts. Such multi-functionality is to be expected when one considers the challenges of life and the parsimony of evolution.

As somebody with keen interest in synthetic biology, I try to take notice of examples of multifunctional bacterial proteins. The fact that a single polypeptide sequence can have many different roles depending on context and environment conditions is an important one to bear in mind if you are attempting to design an artificial protein for a particular function in a synthetic system. The synthetic biologist thus should always be wary of unexpected interactions; ideally, we can mimic nature and try to design proteins that have several synthetic functions.

Even within NusA's known transcription related roles, multi-functionality is manifest. NusA can displace sigma-70 factor from RNA polymerase and promote elongation. In certain genomic contexts, NusA also promotes termination (usually at Rho-independent terminators).Yet at other sequences and scenarios NusA is important for anti-termination of transcription. Moreover, as mentioned by the authors of the study in question, NusA is also thought to be involved in bacterial stress response (through interactions with DinB, a DNA polymerase with certain repair functions) and may act as an RNA chaperone. 

The general concept of multi-functionality aside, real questions remain as to whether or not NusA behaves as a heat-shock chaperone under physiological conditions. While I am of the opinion that the evidence for oligomerization and even chaperone activity in vitro is fairly strong, demonstrating this in vivo is problematic. Indeed, the multi-functional nature of NusA makes it difficult to discern the role it is playing when providing heat-resistance. Furthermore, the authors only demonstrate an in vivo role when over-expressing NusA. This is likely a reflection of some technical difficulty in studying NusA at endogenous levels (Given this, over-expression is an understandable decision during experimental design), but the over-expression may produce misleading results nonetheless.

Pick a gene, any gene. (Is NusA your chaperone?)

Given NusA's canonical roles in transcription, it would be unusual to hypothesis a function as a heat shock 'buffer' chaperone a priori. Why, then, did the authors decide to investigate the behavior or NusA at various temperatures in vitro? I think it is interesting to analyze the various rationale given in the article. I have listed them in bulleted form below:

1. Similarity of the dual repeated acidic domain to other proteins that form oligomeric structures

2. NusA is stable at high temperatures (does not aggregate).

3. NusA fusions has been known to improve the solubility of the fusion partner. Since DnaK and GroEL, known chaperones, also have this effect on fusion proteins, this is taken as evidence that NusA may also be a chaperone.

4. Although not stated in the introduction as a rationale for choosing NusA, the authors mention in the discussion that transcription elongation factor, GreA, also has chaperone activity.

Together, the above points may indeed prompt the hypothesis that NusA may be a oligomerizing chaperone. However, point 3, the concurrence of fusion partners and chaperone activity, is not very convincing. There are several other proteins, such as MBP (Mannose Binding Protein) and GST are used to increase the solubility of fusion proteins. These two proteins, and others, have no known (to my knowledge, at least) chaperone activity. It is curious that this point is given as the final rationale for choosing NusA, since it is arguably the weakest. The demonstrated heat stability of NusA is also a questionable rationalization, since the authors demonstrate that this stability is in fact pH sensitive (NusA is stable at 80 degrees at pH 7.5, but at one pH unit lower it aggregates at temperatures as low as 50 degrees celcius).  

Although questioning the rationale for a study does not really impact upon the strength or important of the work, it can sometimes lead to interesting insights. The inclusion of relatively weak rationalizations in the article may suggest that the NusA was chosen for other reasons, or the oligomerization may have even been a serendipitous observation! It is difficult to tell. Regardless of the motivation for the study, however, the real question lies in whether or not the claims are justified.

A Critical (But Brief!) Review of the Data

Besides the more philosophical commentary and questions raised elsewhere in this article, the data must be examined to validate the claims made by the authors. In some experiments, the most appropriate controls are lacking. For example, in Fig. 2 it would have been useful to use a known sHSP as a positive control, and as a negative control non-NusA protein that does not provide chaperone activity either before or after heat treatment. However, these concerns are answered in part by Figs. S8 and S9; the truncated form of NusA cannot oligomerize and lacks the chaperone activity found in the full length protein.

The most serious limitations in this study are likely a reflection of technical difficulties; the study of NusA in vitro is certainly easier and cleaner than in vivo, but doesn't provide direct evidence of NusA's physiological role as a chaperone. Even the use of His6 tagged NusA has drawbacks, as the endogenous protein does not have this appendage. While it is unlikely that the six amino acid histidine motif has any impact on the results observed (especially considering that the C-terminal domain of NusA appears responsible for the phenotypes), some experiments with native NusA (or His6-NusA with the motif cleaved off after purification, which is not a difficult procedure to execute) would have strengthen this work by eliminating concerns about the role the N-terminus may be playing.

Remaining Questions

This article raises several additional questions. First, it remains to be determined if NusA acts as a stress or heat shock 'buffer' chaperone under physiological conditions. Beyond NusA and it's acidic repeats, what other proteins act as sHSP, and under what conditions? Can systematic investigations reveal a 'chaperome?

Overexpression of NusA, which the authors claim provide resistance to heat stress, is also known to inhibit the growth of a YqgF temperature sensitive mutant (Iwamoto, 2012). What is the molecular mechanism for this phenotype; a direct, or indirect interaction between YqgF and NusA?


Iwamoto., A, (2012) Mutations in the essential Escherichia coli gene yqgF and their effects on transcription. J. Mol Microbiol. Biotechnol. Vol. 22 (1) PMID: 22353788

Kern. R., et al (2003) Chaperone properties of Escherichia coli thioredoxin and thioredoxin reductase. Biochem J. Vol 371, pp. 965-972. PMCID: PMC1223331

Li, K., et al (2012) Transcription Elongation Factor GreA Has Functional Chaperone Activity. PLoS One Vol. 7 (12) PMCID: PMC3521015

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