mvresearch

Problems with the riboswitch assay

2006-10-31T17:05:00.000-08:00

This week I got the preliminary riboswitch experiment results. I tested sxy RNA for binding ATP, GTP, dATP. dGTP, guanosine, adenosine, AMP and GMP (separately). Each of these substances was incubated in three different concentrations (10 nM, 300nM and 100uM) with labeled sxy RNA in a special buffer that contains Mg and K. I also had three controls:

1) RNA incubated with 100uM of ligand and not digested with RNase (negative control)

2) no_ligand control, sxy RNA with no ligand added and digested with RNase

3) ladder control, which is sxy RNA digested with RNase under denaturing conditions so that it cuts all of the C and U residues in the sequence (not only single stranded ones).

The results did not seem very exciting at the first glance, since all of the samples gave the same result. Further investigation revealed the assay is actually not working properly. First, all of the samples gave the exact same bandage pattern and the same intensities of the bands. This would be fine and I would have concluded that the ligand addition is not affecting folding of sxy RNA, if only wasn't for my controls. All of my negative controls were digested in the exact same way as the rest of the samples (these RNAs should be intact since no enzyme was added). The very first thing I suspected was RNase contamination. However, if this has really happened we would expect to see more cleavage in the samples where I added RNase (which is not the case).
Another observation was that all of the samples except for the ladder control had very high background, meaning that there is lot of unspecific cleavage (that gives fragments of various lengths and resolves as darker background in a gel).

One of the explanations of what happened is that the buffer that I used for riboswitch analysis was somehow affecting RNase activity or (and) unspecifically digesting the RNA. The unspecific digestion of RNA usually happens when the solvent is too basic. I checked the pH, concentration and composition of the riboswitch buffer and it is not much different in composition from the regular buffer (the one that I use for secondary structure probing). The only thing that is different is pH - the riboswitch buffer is more basic (pH 7.9) than the regular one (pH 7). According to the technical support that I called, this shouldn’t make much of a difference for RNase activity however, I suspect that this is the major problem in the assay. I did bunch of controls yesterday testing all of the buffers and reagents that I use in the experiment and ran the samples in a gel. I should have the answer of what is going on later today. If it turns out that the riboswitch buffer is the one to blame, I think I will try doing the assay using the regular buffer for riboswitch analysis.

Another news is that I got my thesis back form all of my committee members and I am currently working on the revisions. This shouldn’t take too long and I think that I‘ll have everything done and ready to go by the beginning of next week.

Research status

2006-10-23T09:37:00.000-07:00

In last couple of weeks I handed in my thesis (finally!), I started working on the riboswitch experiment and made a nice poster for the LSI research day.

CHAPTER I - THESIS

I finally finished it!!! The last week of writing was pretty rough: I slept for only couple of hours in the full week. I it took me a full day to finish my table of contents and list of tables and figures (somehow I always thought that those are the stuff that could be done more as a side work). The only thing that my thesis is sill missing is an abstract, which IÂll have to finish this week. Currently, I am waiting to hear back from my committee members.

CHAPTER II - POSTER SESSION

LSI grad research day is taking place tomorrow it seems that is really nicely organized. I am going to present my poster in the afternoon. Actually, the poster is designed to present combined data on regulation sxy expression where we included the results from most of the work on sxy. The poster session starts tomorrow at 330pm. All are welcome!

CHAPTER III - RIBOSWITCHES

This is experiment that I started some time ago. The idea was to test whether sxy mRNA binds small molecules, especially purine bases, nucleosides or nucleotides. Traditionally this is done via in-line probing, which is based on the idea that RNA spontaneously degrades (incubation at 25C in salts) predominantly in single stranded regions. The end labeled RNA is incubated with and without ligands, and resulting fragments are then resolved on sequencing gels. If the RNA binds the ligand the RNA secondary structure changes and this can be detected in the band pattern change.
However, the in-line probing did not work for me and we decided to use alternative way to test sxy RNA by performing nuclease mapping assays. This is similar to the previously described one where the only difference is use of RNases (that specifically cut certain residue type) to degrade RNA.
Since there is so many different ligands that we could test (and both time and money are limited!) I am going to focus on testing nucleotide tri- and mono- phosphates first.
Here is the logic Â H. influenzae cells can gain nucleotides in two ways:

1) Through DNA uptake
where DNA is degraded and the dNTPs are reused

2) and by taking up free nucleotides from their environment. However, cells cannot transport nucleotides unless they are first dephosphorylized to give nucleosides. This happens in periplasm and up taken nucleosides are then converted in nucleotides and used in the cell.

Therefore we would expect that the signal for sxy expression is probably one of the purine nucleotides or nucleosides (this also includes IMP because AMP interconvert with GMP through IMP).

I prepared labeled sxy RNA and ligands that I want to test, and I am going to do the first tests today. This week we should have some interesting (hopefully) results.

Always look on the bright side of life..

2006-10-03T16:36:00.000-07:00

Ok, I am closer to the beginning of the end with my thesis writing.
I admit that it makes me really happy the fact that I have to use an industrial stapler to staple my thesis together. Also the Rosie's comments are getting much shorter and there is more "good"s on the sides of my poor paragraphs.
I think that these are all the signs that I am getting there..

After two sleepless nights, finished my second draft of discussion section today. I am really unhappy with it but I am ready to work on it for the rest of the week knowing that soon it will be completely done. My goal is to finish drafting the thesis by the end of the week. (!!good luck thesis!!)

End now for something completely different-

While I was doing some background reading, (in my pathetic attempt to describe one of the models of sxy regulation in my discussion) I found the data for nucleotide concentrations in the cells of E. coli and it was quite interesting. It turns out that NMPs are far more abundant in the cell than the NTPs, and that GMP is found in much higher concentration in the cell than the rest of the NMPs. Knowing that AMP and GMP can interconvert through a common precursor, IMP and that the conversion is especially important when guanine and adenine compound are present in medium (and available for the cells), the data seems surprising. It would be interesting to find out why this NMPs ratio differs.

Does anyone have an explanation?

Very first thesis draft

2006-09-26T09:44:00.001-07:00

Last week I have been working on writing of the discussion section and preparation of my first thesis draft. The discussion section is still not fully developed but I got a very basic first draft.

Additionally, it was my turn last week for lab meeting, so I turned it into discussion on my thesis discussion. I presented some ideas on the regulation of sxy genes. Based on the information that we have so far I proposed that sxy could be regulated by one of these three mechanisms: transcription/ translation coupling, action of a riboswitch or by binding of non-coding RNA. At this point it seems that sxy regulation can be explained by each of these mechanisms however, there are also some problems and none of this models fits perfectly into the picture. I think that I’ll still have to do some reading on this subject (and maybe to discuss it with my lab mates:)).

Yesterday I revised the intro and methods sections and I think that they got much better. Today, I am starting with the revision of my results section (which at this stage has quite a bit of problems). The goal for this week is to have a nice readable draft with all of the figures and references included.

discussion on my thesis discussion

2006-09-18T18:22:00.001-07:00

Last week I finally finished the first draft of the introduction and handed it to Rosie. I am still not very happy with it but at least it is a start and I'll work from that.
I think that I have a problem to make a distinction between important stuff and details. Additionally, I find it really hard to make up a story that is interesting and easy-to-follow (than again, maybe science is not supposed to be told in an interesting way?). They say it comes with time and practice, but I do not have either.

Today I started working on the discussion section. I made a very rough outline and realized that I 'll have quite a bit of reading to do to be able to propose the regulation of sxy gene. I have some ideas how to organize this section and what kind of arguments to make.

Tomorrow is my turn to give a lab talk, so I'll have a busy night preparing the presentation on my discussion (which I still do not have). I am kind of looking forward the presentation, hoping that I'll get feedback that will improve my analysis and give me new ideas.

still working on the gel picture........

2006-09-12T18:45:00.000-07:00

I ran some gels last week and it turned out that the label (on the RNA) was really old so I had to expose them for days and days to be able to visualize the bands (and they were still fuzzy). I ran another gel today and I will have to leave for at least 4 days to get something (I am getting really annoyed with it).

I finished the first version of the gel figure that we want to include in the sxy manuscript. I decided that it is not the_worst_thing_ ever to crop your gel picture and to show only the lanes that carry important info. It is a bit of a patchwork, but I think that it doesn't look that bad.

Thesis writing is going very slowly. I have to do some background reading to improve my introduction info. Hopefully, I will have this section done (together with Methods and Results section) in couple of days. Then, I can finally start working on the discussion!!

Back to lab

2006-09-07T10:47:00.003-07:00

After couple of weeks of sitting at my desk and trying to write my thesis, I decided to run more sequencing gels. Why? - you ask.

Our lab is currently preparing a manuscript on regulation of sxy gene in H. influenzae and we would like to present the data showing that sxy RNA secondary structure limits the sxy expression.
One way of indirectly showing this is to do the mapping of the secondary structures of RNAs in the wt sxy and of RNAs of sxy hypercompetent and non (or hypo) competent mutants. Both of these mutants carry point mutations that affect the stability of their RNA secondary structures.

The analyses that I did so far confirm that the secondary structure of sxy RNA is less stabile in the hypercompetent mutant and more stabile the hypocompetent mutant. Eventhough I got nice and reproducible results (with nuclease mapping of secondary structures), I still do not have the one -? perfect and publishable picture.

This week I will try to produce a nice gel picture showing the differences in the sxy RNA secondary structure in these different RNA species. Although this sounds as a really simple thing to do, I can easily assure you is highly stressful procedure!
Yesterday I prepared the RNA samples and today I am running them in a gel. Hopefully we'll get some very nice results tomorrow!!

Introductory week

2006-08-29T17:32:00.001-07:00

***this was supposed to be posted on Tuesday but for some strange reason my blogger saved it as a draft***

This week is a week of my thesis introduction. I already have detailed outline of this section but I really need to start working on putting up some paragraphs. I'll also have to read a bunch of papers in order to be able to give good background info for the projects that I did.

The results section is still not completely done! I am working on writing up the results for my bionformatics project that I did some time ago (but it really takes time to get back into those thoughts). I also had to redo some analysis for the portion of this project - I wanted to make sure that the data I am presenting in my thesis is correct.

The more I think about this project the more I find it interesting.
The bioinformatics project was done to test whether the secondary structures of sxy RNA in Pasteurellaceae are similar (our famous H. influenzae is a member of this family). The hypothesis was that the RNAs in sxy homologues would fold in similar manner if the secondary structure were the one responsible for regulation of this gene.

Eventhough the analysis suggested that there is no common secondary structure of sxy RNA in this family, it showed that the intergenic regions upstream of this gene are quite long (~600 nt). This implies that a regulatory element (of some kind) could be present in the region just upstream of sxy (perhaps in the untranslated regions of their RNAs). This number (600 nt) is even more striking when compared to the mean length of intergenic regions in E. coli (I think that it is about 55nt ).

Unfortunaly, we have no experimental data of the RNA of sxy homologues in other Pasteurellaceae, (we are not sure if they even have untranslated regions in their RNAs), but as other things in science, it make us wonder and think of new ways to test these interesting questions.

Another day in science....

2006-08-24T17:14:00.000-07:00

I could summarize my last couple of days at work in two words: Thesis Writing.
No experiments,

no analysis

– just simple and boring - thesis writing!

I have a feeling that all of the people in the building are sick of me when I start talking about my endless writing of M&M and Result section.
This week I remembered how stressful writing can really be. The fact that I am behind my schedule really does not help either. This week I wanted to start with my introduction section but that fell into the water...

For some non-scientific stuff - if you have some time left for not thinking about science go to see the inconvenient truth. I think the movie is good in showing the facts about global worming in general (it is really striking to hear all those facts). It also gives loads of suggestions on how to act to make our planet better place to live (this sounded really geeky).

Wriiiiiiiiiiiiiiiiiiiiiting

2006-08-21T20:51:00.000-07:00

I have been working on my thesis writing for some time now, which is not much of a fun. I am still stuck with my Method and Results section, mostly working on editing, editing and editing....
I think I am finally done with most of the figures that I want to put into my thesis (at least for the sections that I just mentioned).
BTW, I heard yesterday that there is a guy at UBC that just finished his thesis writing in a week (!!!!) - I would love to know how he did it!

For my riboswitch experiment - I got even more confused!
Last week I repeated the experiment of sxy RNA binding adenine (with different concentrations) and ran the samples in a gel on Saturday.
Finally, I got my answer yesterday - the patterns were all the same for both all of the different adenine concentrations and the negative control (and this is quite different form what I got the last time). All that I can conclude from this is that we need to do more repetitions and that the conditions (for the sample preparation) should be kept strictly constant.

To summarize, what needs to be done regarding the riboswitch experiment follows:

1) Repeat the experiments adding the same ligands at same concentrations that I used

2) Use nucleases that I still haven’t tested (TI and VI) -
but being aware of incompatibilities between RNase TI (specific in cleaving ssGs) and guanine (or its chemical “relatives”)

3) Getting a negative and a positive control
-Finding a RNA (that we know that it doesn't bind purines or other ligands to be tested) that has a similar length and testing for binding

-Getting a known riboswitch sequence and testing it for binding a specific ligand.
Doing these experiments would make us pretty sure that the assay is working and that it is sensitive enough for testing sxy RNA.

This Week's Science

2006-08-17T21:17:00.000-07:00

For the last couple of days I have been working on couple of things. Here is a short list:

1) Exciting riboswitch experiments.

I have been testing sxy RNA for ligand binding by adding different ligands and then doing nuclease probing by RNase A to check if a conformational change will happen. Since I was not sure if the RNase assay will work due to the change of reaction conditions (from my regular nuclease conditions), I initially tried it just by adding three different amounts of adenine (as ligand). The gels that I ran gave really interesting results.
First, they confirmed that RNases can happily work in almost any conditions and second, the cleavage pattern was different when the different concentration of ligand was applied. However, I am not 100% convinced that this is simply a reaction to ligand addition: the samples with same adenine concentrations partially digested with different enzyme amounts gave a bit altered pattern.
I repeated the experiment testing for binding of xantine, guanine and hypoxantine, then ran the samples in a gel. The results showed the effect on ligand addition is present in samples that had either guanine or hypoxantine added Cleavage pattern for xantine was same for all of the tested concentrations.
To test if this is a ligand_binding_ effect only, I repeated the experiment with adding different adenine concentrations. If the assay is good I expect to get the same result as for the first trial when the same conditions were applied. I ran a gel with this samples on Wednesday and I'll get the results tomorrow.

I am planning to repeat the experiments using same ligands but different enzyme (RNase TI) to check if the effect will be the same when the same amounts of ligand is added.

2) Thesis writing.

Ok, I started panicking about my writing, so that is why I spent most of the time this week in the office, typing. Today I finally finished the first draft of my Methods and Results section. I am sure that there still going to be lots of work to polish this portion up but I think that this is a good start.

3) Racking tips and media making.

....Officially, I started having dreams about tip racking...
I usually don't mind doing the lab staff, I think, however, that the tip racking is the most boring of all lab activities.

Ribowitch day

2006-08-11T18:31:00.000-07:00

I created a new way to test if the RNA that I am testing is responding to addition of ligands by changing its conformation. The last method that I was using wasn't giving me any results, so we decaded to change it a little bit. Yesturday I ran my samples in this newly prepared way and today I found out that the assay is working fine.

Today was a riboswitch day since I prepared even more samples, testing if the RNA is binding any purine base or their chemical 'relatives (like xantine and hypoxantine). It was a tidious and long process but I hope we will get some nice results. Everithing would seem so much easier to explain if sxy was regulated by a riboswitch.

To flavour my day, I also worked on my thesis writing. The end is still long way ahead....

my very first blog posting

2006-08-10T17:49:00.000-07:00

This supposed to be my research blog so I'll talk about the staff that I am currently working on.

Ok, I am interested in how this gene (called sxy) found in Haemophilus influenzae and most of gamma proteobacteria is regulated. We know that sxy is necessary for the development of natural competence (the process that allows bacteria to �eat� DNA molecules from their natural environment, and sometimes, this allows incorporation of the DNA parts into their genomes.

We also know that sxy is probably regulated at the RNA level:
1) my experimental analyses (using nuclease RNA secondary structure mapping) have shown that 5� end of sxy mRNA folds into complex stem-loop structure,
2) introduction of a single mismatch in the stem portion of the structure dramatically changes sxy abundance in the cell implying that the stem is particularly important for the regulation of the gene.

Another thing that we know about sxy is that the addition of purines and their derivatives changes its expression. This interesting fact led to the idea that sxy could be riboswitch regulated. These newly discovered gene cis-regulators are found in the untranslated regions of mRNA and fold into a very conserved structure. Part of this structure usually serves as a receptor, binding a specific ligand (usually small molecules like amino acids, or vitamins) and changing the original conformation of the molecule.

So..
........these days I�ve been testing if sxy is riboswitch regulated. I am doing this by probing the RNA secondary structure using RNases. Basically, I would just fold end labeled sxy mRNA in vitro, add a ligand (in different concentrations) to some of the samples and than partially digest it with RNases. After resolving the fragments in a gel the change in the cleavage pattern will show if the RNA is responding to the ligand addition.

Where I am right now? I just prepared my RNA samples by exposing them to different concentrations of adenine, digested them and ran the samples in a gel. Gel exposing usually takes a day. So - tomorrow the results will be uncovered....