How do scientists measure protein production productivity? Here are some of the methods you might see – and I saw a lot of them today at CSHL’s annual In-House Symposium, where professors from all different areas of research give talks on what they’ve been up to – and I successfully completed my session-chairly duties! Huge thanks to the speakers for sticking to the allotted time so I didn’t have to tell big-wig scientists to stop talking!
Making a protein’s kinda like a cellular version of baking a cake. But the ingredients are molecules called amino acids and, instead of tasting yummy but rotting your teeth, the products do things like build other molecules, break down molecules, relay messages, and keep you alive. Because they’re so important, scientists often want to know how “popular” certain recipes are. And we can MEASURE this GENE EXPRESSION in multiple ways that tell us about different parts in the process – how many copies of the recipe are made? are those copies still in circulation? are they actually being used? are the final products sticking around?
Each protein has its own “recipe” in the form of a GENE. The gene’s written in DNA letters and housed in a “library of cookbooks.” a membrane-bound compartment in each cell called the nucleus. You can’t just “check things out” from the Library of Congress, and you can’t check genes out from the nucleus. These genes are precious because they hold the instructions for making you. So you don’t want to tamper with the originals. Instead, if you want to use one, you first must make an RNA copy in a process called TRANSCRIPTION.
This copy gets processed into mature messenger RNA (mRNA) that acts as a messenger to tell the recipe to the “bakers” – protein/RNA complexes called RIBOSOMES. To make a protein, the ribosomes travel along the mRNA “calling out” for amino acid “ingredients” that molecules called transfer RNAs (tRNAs) bring to them to get added to the growing chain. This process is called TRANSLATION.
Unlike cakes, which are single-use, proteins usually are designed to be used multiple times. They’re used rather than consumed. But they can get “thrown out” if desired. Unwanted proteins are tagged with a chain of a little protein called ubiquitin, which a protein version of a paper shredder called the PROTEASOME shreds so you can recycle the parts.
So, the amount of any protein in a cell at a given point in time depends on how many copies were made (transcription level); how many copies still exist (taking into account mRNA degradation); how many copies are being used; how many bakers are using them (ribosomal density); how quickly the bakers bake (translational efficiency) and how quickly they’re being degraded (protein degradation). There are different ways to look at these various aspects (only some of which I’ll mention), starting with
How many copies are being made? -> you can use Pol II CHIP-seq to look for enrichment of RNA polymerase (the DNA->RNA Xerox machine) around the gene to get a relative sense. Basically, you “freeze” RNA polymerase (the DNA->RNA Xerox machine) in place by cross-linking it to the DNA it’s bound to. Then you isolate the DNA-bound RNA pol & see where it’s bound. This gives you a sense of the recipe’s popularity in terms of transcription.
How many copies are still in cellular circulation? mRNA can get degraded, so if you want to know how many copies are still in a cell, you’ll want to measure that specifically.
And speaking of degradation, RNA is less stable than DNA, so, before you put your data in danger, you’ll want to convert the RNA into DNA form. We call this DNA copy cDNA. It differs from the original genomic DNA (gDNA) form of the gene in that it’s the edited form – it has the introns removed. Since you’re going in the reverse direction from transcription (which makes an RNA copy of a DNA gene) we call this REVERSE TRANSCRIPTION.
There’s a lot of RNA – and DNA – but you only want to look for mRNA. So you need some generic mRNA feature. One thing they all have is a poly(A) tail. And what binds poly(A)? Poly(T)! So you use a short stretch of Ts (but in the deoxyribose form cuz we want to make DNA) -> oligo-dT as a primer. This tells the reverse transcriptase where to start copying.
Once you have a more stable version, you can move on to the quantitative PCR part of RT-qPCR. Use specific sequences that are unique to specific recipes to make more copies. The amount of copies you end up with depends on how many copies you started with, so you can compare to recipes with fairly constant levels.
This is good if you only have a few recipes you’re interested. If you have a lot you can use a microarray, which is like a plate with lots of wells and inside each is a miniature Northern Blot. What’s a Northern Blot? Glad you asked.
A Northern Blot is a way to go RNA fishing. for “bait” you use a complementary piece of DNA that you label (usually with a radioactive phosphate). First, you separate the RNA fragments by size by running them through an agarose gel – the gel matrix is like a mesh that slows down bigger fragments more cuz they get tangled up in the mesh as they try to squeeze through. Once you’ve separated them you need to transfer them to something more stable before they diffuse out or you tear the gel. So you transfer them to a nylon membrane (like a durable piece of paper that nucleic acids like to stick to). You do this by applying charge in the vertical direction (not horizontal like the gel was). And then you see if the matching DNA sticks and how much.
If you’re interested in “all” the recipes, you can use “next gen” RNA-seq. You isolate the RNA and use random primers to make lots of copies of whatever’s there so there’s enough to see.
Of course, if you rely on these mRNA-level measurements, you’re assuming that all of the measured mRNA gets turned into functional bakeries (mRNPs) that crank out lots of baked goods (proteins). And that those proteins last. And that may not be the case.
So let’s look one step further. Measure how many copies are getting turned into functional bakeries – in order to form a functional protein-producing “bakery” you need certain proteins to bind it to help get things up and running and help the bakers as they travel along the recipe and piece together the corresponding amino acids.
POLYSOME PROFILING looks at whether recipes are associating with full ribosomes and how many. The ribosome has lots of parts, but it has 2 main “pre-fab” “halves” – a small subunit & a large subunit. It needs both to be functional. Because the halves aren’t really halves, we can tell them apart by their weight. -> Separate them in a sugar gradient. The bigger half is denser so it will “sink” further. Both together will sink even further
And a cool thing is that the halves are “glued together” by mRNA binding – they don’t normally associate. So an “intact” ribosome implies there’s a recipe poised to be baked. A single (mono) full ribosome on an mRNA is called a MONOSOME. But usually, active bakeries have lots of bakers – POLYSOMES are multiple (poly) ribosomes attached to a single mRNA. And they weigh even more than the monosomes. So they’ll sink further.
You can detect “global” differences if the ratios are skewed or you can look to see where certain recipes end up by doing a Northern Blot on the various fractions. Since different mRNAs are different lengths, and the longer the length, the more ribosomes can be bound at a time (but the longer it will take each to finish) you can take this into account – look at ribosomes per length unit when comparing
How much protein are you ending up with?
You can look at “all” proteins – if you hear “all” think “-omics”! Hear “-omics” think “big data.” The -omics folks use computer wizardry to look at big data sets and find trends, etc. Genomics looks at DNA, transcriptomics looks at mRNA, proteomics looks at proteins.
Mass spectrometry (mass-spec) splits proteins into small charged (ionic) fragments then matches those fragments to the possible proteins they came from to figure out what proteins were in a mixture.
If you want to look at specific proteins, you can use a Western Blot. It’s like the Northern Blot except you run a different type of gel (made of polyacrylamide, not agarose). And you use a different type of probe – proteins don’t have “complementary sequences” like RNA & DNA do, so you use something that recognizes some specific site on the protein (similar to how drugs can bind pockets on proteins, but in this case you use little proteins called antibodies – different ones recognize different proteins. And you use a membrane that binds proteins.
The SDS-PAGE gels are run vertically and then you transfer it horizontally. Then you can “go protein fishing” – for “bait” you use an antibody – a small protein that binds specifically to other things. You use antibodies specific to certain parts of specific proteins. You put the membrane in a bath filled with that antibody to let it bind, then see how much bound. (there’s a lot of washing and blocking and stuff to make sure that you’re only detecting legit binding – they’re a real pain…)
But wait – what if the protein’s being made, it’s just not lasting long enough to detect? What if it’s all getting shredded by the proteasome? You can do the experiment with and without a proteasome inhibitor to see if protein’s being made but destroyed.
there are other ways to measure various things, but these are some of the main ones.