Thursday, June 11, 2015

Biochemists can be astronauts!

The latest issue of ASBMS Today has an article about the American astronaut Paggy Whitson [see A lab with a view].

Peggy Whitson is a biochemist. She did her Ph.D. with Kathleen Mathews at Rice University in Houston, Texas, USA. I frequently refer to her work on the lac repressor and its interaction with lac operator sequences [see Repression of the lac Operon]. Here are some of her papers. Once you understand this stuff, you are in a better position to judge the ENCODE results and the role of spurious binding sites.

Hsieh, W.T., Whitson, P.A., Matthews, K.S., and Wells, R.D. (1987) Influence of sequence and distance between two operators on interaction with the lac repressor. Journal of Biological Chemistry, 262: 14583-14591.
The influence of additional operator or pseudooperator sequences on the lactose repressor-operator interaction has been investigated. Results of kinetic and equilibrium binding measurements suggest an important in vivo role for the Z-gene pseudooperator in repressor-operator binding; the formation of a ternary, looped complex is indicated by the influence of secondary operator sites on binding parameters. Although the binding affinity of the Z-gene pseudooperator [Oz] is only approximately 1/30 that observed for the primary operator [O], the binding affinity to DNA containing both Oz and O is significantly higher than either sequence alone or the sum of the two. This synergistic effect is enhanced further by replacing the pseudooperator sequence [Oz] with the primary operator sequence and results in an even stronger ternary complex in plasmids with duplicate primary sites. The distance between the center of the two primary operators affects the formation of a ternary complex in the linear DNA molecules. Decreased dissociation rate constants were observed with spacing of operator-like sequences between 300 and 500 base pairs (bp). Minimal influence of the second operator on repressor binding is observed when the operators are separated by approximately 4000 or approximately 100 bp. The significant influence of distance on kinetic and equilibrium parameters was demonstrated by measurements on plasmid pRW1511 [Oi-O-PL-Oz] cleaved with restriction enzymes either in the polylinker region to place Oi-O and Oz on opposite ends of the linear plasmid or outside this region to maintain the sites within 500 bp. These results are consistent with the formation of operator-repressor-pseudooperator ternary complex to generate a looped DNA structure.

Whitson, P.A., Hsieh, W.T., Wells, R.D., and Matthews, K.S. (1987) Supercoiling facilitates lac operator-repressor-pseudooperator interactions. Journal of Biological Chemistry, 262:4943-4946.
The binding affinity of the Escherichia coli lactose repressor to operator-containing plasmids was increased by negative supercoiling of the DNA. The increased affinities observed were dependent on the sequence context of the DNA as well as the degree of supercoiling. Dissociation rate constants for plasmids containing a single operator site decreased as a function of the negative supercoil density. However, the presence of pseudooperators in the plasmid DNA in addition to the primary operator sequence resulted in a significant decrease in the operator-plasmid dissociation rate at higher negative supercoil densities. Approximately eight ionic interactions were determined for both the supercoiled plasmids and the linear DNAs examined. These results suggest that the stabilization provided by the topology of supercoiled DNA affects the nonionic component of the protein-DNA interaction. The ability to form a ternary complex of protein with two DNA segments is increased by the presence of multiple operator-like sites on the DNA. Furthermore, supercoiling DNA with multiple operator-like sequences profoundly diminishes the dissociation rate and results in a remarkably stable ternary, presumably looped complex (t1/2 approximately 28 h). These data suggest a critical role in vivo for DNA topology and pseudooperator(s) in transcriptional regulation of the lac operon.

Whitson, P.A., Hsieh, W.T., Wells, R.D., and Matthews, K.S. (1987) Influence of supercoiling and sequence context on operator DNA binding with lac repressor. Journal of Biological Chemistry, 262(30), 14592-14599.
The dissociation of the repressor-operator complex from a series of negatively supercoiled plasmid DNAs was examined as a function of the sequence context, orientation, and spacing. The plasmids were grouped into four classes, each with common sequence context. The highest dissociation rate constants were observed for the plasmids containing only a single operator (or pseudooperator) sequence, while approximately 10-fold lower rate constants were measured for plasmids with the I gene pseudooperator in conjunction with either the Z gene pseudooperator or the primary operator. Comparison of the behavior of these two classes of plasmids demonstrated the importance of two operator sequences and supported a model of DNA loop formation to stabilize the repressor-operator complex (Whitson, P. A., and Matthews, K. S. (1986) Biochemistry 25, 3845-3852; Whitson, P. A., Olson, J. S., and Matthews, K. S. (1986) Biochemistry 25, 3852-3858; Whitson, P. A., Hsieh, W. T., Wells, R. D., and Matthews, K. S. (1987) J. Biol. Chem. 262, 4943-4946; Krämer, H., Niemöller, M., Amouyal, M., Revet, B., von Wilcken-Bergmann, B., and Müller-Hill, B. (1987) EMBO J. 6, 1481-1491). The third class, with intermediate dissociation rate constants, was comprised of plasmids which contained the primary operator and the higher affinity pseudooperator normally located in the Z gene. Neither the additional presence of the I gene pseudooperator nor the orientation of the primary operator relative to the Z gene pseudooperator significantly affected the dissociation rate constants. The binding characteristics of this group of plasmids demonstrated the essential role of the Z gene pseudooperator in the formation of intramolecular ternary complex and suggested an in vivo function for this pseudooperator. Plasmids containing two primary operator sequences were the class with lowest dissociation rate constants from lac repressor, and minimal effects of salt or spacing on dissociation of this class were observed. These data are consistent with formation of an intramolecular complex with a looped DNA segment stabilized by the combination of increased local concentration of binding sites and torsional stresses on the DNA which favor binding in supercoiled DNA.

Whitson, P.A., and Matthews, K.S. (1986) Dissociation of the lactose repressor-operator DNA complex: Effects of size and sequence context of operator-containing DNA. Biochemistry, 25:3845-3852.
The dissociation kinetics for repressor-32P-labeled operator DNA have been examined by adding unlabeled operator DNA to trap released repressor or by adding a small volume of concentrated salt solution to shift the Kd of repressor-operator interaction. The dissociation rate constant for pLA 322-8, an operator-containing derivative of pBR 322, was 2.4 × 10-3 s-1 in 0.15 M KCl. The dissociation rate constant at 0.15 M KC1 for both Xplac and pIQ, each of which contain two pseudooperator sequences, was ~6 × l0-4 s-1. Elimination of Elimination flanking nonspecific DNA sequences by use of a 40 base pair operator-containing DNA fragment yielded a dissociation rate constant of 9.3 × 10-3 s-l. The size and salt dependences of the rate constants suggest that dissociation occurs as a multistep process. The data for all the DNAs examined are consistent with a sliding mechanism of facilitated diffusion to/from the operator site. The ability to form a ternary complex of two operators per repressor, determined by stoichiometry measurements, and the diminished dissociation rates in the presence of intramolecular nonspecific and pseudooperator DNA sites suggest the formation of an intramolecular ternary complex. The salt dependence of the dissociation rate constant for pLA 322-8 at high salt concentrations converges with that for a 40 base pair operator. The similarity in dissociation rate constants for pLA 322-8 and a 40 base pair operator fragment under these conditions indicates a common dissociation mechanism from a primary operator site on the repressor.


12 comments :

  1. Larry,

    You definitely don't know the inside scoop. THEY are sending a biochemist with the obvious hopes and a lot of specimens in tubes. THEY hope life can start in space provided it has the right environment. Scientists can't do it in the lab on the Earth but they have high hopes that dumb luck will be lucky this time. How can you go wrong? It no more than a lottery... this time...

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    1. Get your meds Pest, you're blathering again.

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    2. KevNick,

      I would expect you not to read the post properly. But I never expected it to be so obvious that you did not read beyond the title.

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    3. There's a reason why people like KevNick are called IDiots.

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  2. Larry, what is your personal opinion on manned space missions? A scientific necessity or a waste of good (terrestrial-based) grant money? Are there real tangible benefits for studying biochemistry in microgravity?

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  3. But, but, but those papers were written in the 80s, the 1980s!

    Sadly, I do find that many US trainees are severely deficient in the literature and are actively trained by mentors to read specifically within their narrow fields. I expect because during the NIH doubling the rapid and massive increase in biomedical faculty (of which I am one) the emphasis in hiring was on who published the most papers in the fancy journals (of which I was not one). There seems to be little importance put on breadth and ability to span multiple fields in many laboratories these days.

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    1. "are actively trained by mentors to read specifically within their narrow fields"

      If only that was true. But they seem trained to read only a few words of a few papers that might be related to their specific fields.

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  4. And what biochemistry experiments will she be doing when she gets to the ISS?

    Something called ‘GeneLab’ apparently:

    http://genelab.nasa.gov/

    “GeneLab will gather spaceflight genomic data, RNA and protein expression, and metabolic profiles, interface with existing databases for expanded research, offer tools to conduct data analysis, and create a place online where scientists, researchers, teachers and students can connect with their peers, share their results, and communicate with NASA…….. The experiments managed by GeneLab are expected to generate vast amounts of molecular data, which will help define the transcriptome, proteome, metabolome and fundamental genomic responses of a variety of organisms to spaceflight……. GeneLab will create an open access data repository for scientists to perform more research, using a big data, systems biology approach to access the enormous amounts of raw data GeneLab generates. The raw data will come from mapping the complete genes, transciptomes, metabolomes and proteomes of the tissues flown in space. The data will be uploaded into the GeneLab bioinformatics database, a universally available life sciences database that will contain the integrated gene and biomolecular maps for the tissues and organisms that have flown aboard the station.”

    Sounds like a dreadful ENCODE-style, hypothesis-free, data-dredge.

    ‘Omics-in-space’ FFS.

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  5. Oh dear. It's all part of the notion that systems biology means generating vast amounts of data, whereas it should mean trying to understand how systems wrk.

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  6. How come I have this image in my head of Larry in a long robe, holding a light sabre, Going up against the emperor (Barry Arrington) and his pet Sith Lord (Gordon Mullings)?

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    Replies
    1. That's the wrong image. Imagine me with a shotgun, ... and fish, ... and a barrel.

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    2. Watch out. Gordon (KairosFocus) Mullings might use your statement above as a threat of violence against him.

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