Science Buddies: "Ask an Expert"

Hi all,
This probably sounds like a stupid question (and yes, despite what teachers insist upon, there indeed are stupid questions), but I was reading a journal article, and it suddenly occurred to me that a nucleotide (nt) is only half a base pair (bp). So when the article states "As reported by others, the size distribution of S. cerevisiae introns has a distinct bimodal pattern, with approximately 25% of the S. cerevisiae introns falling in the size range of 401 to 2,000 nt" ... is it safe to rewrite this information in my background paper as "... introns fall in the size range of approximately 200 to 1000 bp"? I wanted to keep the unit of measure consistent in my paper, so I wanted to make sure that the conversion from nucleuotides to base pairs was legit.

Also, another question concerning the scientific lingo-- it's not simply the definitions that are giving me trouble-- I can and have looked those up... it's the which-affects-which problem that I'm having here...
Could somebody please help me distinguish between splicing efficiency and transcription frequency and gene expression? My impression of the connection between these three terms are that:
1. transcription frequency is how often a gene is transcribed
2. splicing efficiency is how well a gene is spliced out.
3. gene expression (level) is how much protein product is produced from the gene after translation.
I need to figure out what happens when there is a low splicing efficiency. What happens if there is a "low" splicing efficiency? I don't understand this, because I had always thought that either a gene was spliced out, or it wasn't. (of course, in science nothing is ever as clear-cut as I would like it to be..) Does it affect gene expression? Does that mean that the intron stays in the genomic sequence, or that the gene will be mutated or that the protein product will be altered? Sorry, I'm just listing some possibilities that I can imagine. And how does transcription frequency tie into all of this? I was looking at the Yeast Intron Database, and that was a set of data they had.

I understand if you cannot address all the parts of my question, but I'd really appreciate it if you could help me understand as much as possible.

Thank you for your help! : )
-M

Methionine,

It is true that a nucleotide is only half a base pair. You will probably want to check this with another Expert, but my guess is that the researchers used "nt" rather than "bp" because they had split the DNA fragment to sequence it. From the article (I found Kupfer et al. 2004; is that your article?), I think it is safe to equate nt with bp. (25% of the introns fall in the size range of 400-2000 bp.)

Realize also while you are writing your background information for the article that every number is important. The researchers say that 25% of the introns are between 400 and 2000 bp; the remaining 75% are <400 or >2000 bp.

Transcription frequency is how often a gene is transcribed. It depends on transcriptional controls such as whether the regulatory gene is turned on or off, how tightly the DNA is wound in the area, whether certain proteins called transcription factors have bound to the DNA.

Splicing is a posttranscriptional control-- Not all parts of the resulting mRNA code for a protein, so these must be cut out. The same protein can result from different splicing patterns on different mRNA fragments; or different proteins can result from different splicing patterns on the same mRNA fragment. I imagine that splicing efficiency tells how many of the introns that need to be cut out are indeed cut out.

Gene expression is the amount of protein produced. It depends on posttranscriptional controls (how quickly the mRNA exits the nucleus and reaches the rRNA), translational controls (whether the mRNA is stabilized by a hormone), and posttranslational controls (protein folding, protein activation, feedback inhibition, for example).

A low splicing efficiency I take to mean that either not enough introns were cut out, or some of the exons were cut out in addition to the introns. Either way, the mRNA chain will be different and so will the polypeptide chain created during translation. Whether or not the resulting protein was expressed would depend on posttranslational controls.

I'm sure you are already familiar with a lot of this from your reading, but I hope that going over it in terms of what controls occur when will help you understand exactly what is going on.

Science is rarely as clear-cut as any of us would like, but that can be the fun of working with it.

Sonia

Hi Sonia,
Thanks for your reply. Yes, Kupfer et al. is the article I was reading.

In your reply, you wrote:

Gene expression is the amount of protein produced. It depends on posttranscriptional controls (how quickly the mRNA exits the nucleus and reaches the rRNA), translational controls (whether the mRNA is stabilized by a hormone), and posttranslational controls (protein folding, protein activation, feedback inhibition, for example).

This is slightly irrelevant to my initial question, but all those things you listed about posttranslational controls-- Does that mean that aside from the fact that each gene is probably regulated in a slightly different way (ex: some use feedback inhibition, while some do not and rely on protein folding to get the desired effect/expression), that when I analyze my expression results, I will somehow have to take into account the normal known method of regulation of my gene of interest? I'll clarify -- Because splicing is a form of posttranscriptional control as you said, and posttranslational controls are the final thing that affects gene expression, I would have to take that into account when I analyze my results. Is this correct? Or will that be taken care of by utilizing controls in my experiment? ... This is my first project and I am still trying to fully grasp the concept of "controls" here.

If it helps in answering my questions, my project is about the effect of intron length on degree of gene expression.

Thanks,
-M

Methionine,

Let me clarify the posttranslational controls a little. Translation only creates a polypeptide chain; this must be folded into a functional protein. Feedback inhibition is one way the cell knows that no more of that protein needs to be made-- the final product binds to an enzyme and prevents it from making more of the protein.

I think that all DNA on bacterial plasmids is translated into proteins, so if you are working with those, you would not need to worry so much about posttranslational controls. If you are working directly from a bacterium's main DNA loop or with a eukayotic cell, posttranslational controls could alter your results. I'm not an expert in this area, however. You might like to discuss this with your teacher or mentor.

Experimental controls are parts of your experiment that are not manipulated. They give you a basis for comparison for the variables and tell you that your methods are sound. Here is a simple example: If you are treating bacteria with different substances (to see what will kill them, for instance), one set of bacteria will get no treatment. This is your control. You can compare the other treatments with the "no-treatment" set to see how effective they were. Also, if no bacteria show up in the "no-treatment" set, you know that something went wrong in your procedure.

If you are still having trouble with experimental controls and are willing to give me more details on your project, I might be able to help you see what the controls are in your experiment.

Sonia