But now, on to brass tax. If you’re reading this, it’s because you either want or need to learn to use BLAST, the genetic Google hosted by the National Institutes of Health. This article is going to focus on searching if you already have an amino acid sequence, NOT A NUCLEOTIDE SEQUENCE. So, without further adieu, let’s go!

Step 1: Verification

So, the first thing you’ll need to do is to verify that you have an amino acid sequence rather than a nucleotide sequence. This is probably going to be the easiest part for those of you who are just getting started with BLAST. Your sequence is going to be a text file, just like any other research paper that you’ve ever written or read. But it’s going to be a bunch of unreadable letters. You can tell the difference between an amino acid sequence and a nucleotide sequence by just glancing at the letters you see.

Nucleotide

ATTGCGTTCGAGTCACTATGTATGGCCTCCACGGTAGGTTGAGCAGTACC
TGGCGGTATGACCACCTCCTCAGCGACGATGCTTATGGAGGCGCTGGACA
AGCGTTGACCCAGAGCTTTGGTCCCCAGAGCAAGAAGACCACTGGCCCGA
CACAAGAACACTTCCTCCTTTCCATTAGGGTTCGAGAATAAAGCTATCAG
CTGAGTCAATGCATTGCCACTTTTGAGTCCTCAAGCTAGATAAGTCTCCC
TTTTAAGAAACGCACGAGTACGCCTCTCTAGCGGTTTCTCATCGGACAGC
TCCTACGAAAGCGATCTTTATCGGGATCCACCGACTGTCGGCCTACAAGG
TGGGCCTTTTTGGACCACCCCGAGTAGATCGGCGACCTTTCTTTGTATGC
CAATTCATGAGTAACCTGAGCAGATTGAATGTACACGCAAAATGTCGATC
TAAGTGTCCCGTCCAAGAAGAATTTTTTCTTACTACCCCAGCCTGGTTTA

Notice how this is all A, T, G, and C? It’s a nucleotide sequence of a nucleic acid (which is made up of A, T, G, and C… so it’s DNA. But you already knew that.) (…even if you didn’t realize that you knew it…)

This isn’t what we’re looking for this time. We want a protein (amino acid) sequence.

Amino Acid

MDPHNPIVLDQGTGFVKIGRAGENFPDYTFPSIVGRPILRAEERASVATPLKDIMIGDEA
SEVRSYLQISYPMENGIIKNWTDMELLWDYAFFEQMKLPSTSNGKILLTEPPMNPLKNRE
KMCEVMFEKYDFGGVYVAIQAVLALYAQGLSSGVVVDSGDGVTHIVPVYESVVLSHLTRR
LDVAGRDVTRHLIDLLSRRGYAFNRTADFETVRQIKEKLCYVSYDLDLDTKLARETTALV
ESYELPDGRTIKVGQERFEAPECLFQPGLVDVEQPGVGELLFNTVQSADVDIRSSLYKAI
VLSGGSSMYPGLPSRLEKELKQLWFSRVLHNDPSRLDKFKVRIEDPPRRKHMVFIGGAVL
ASIMADKDHMWLSKQEWQESGPSAMTKFGPR*

BINGO! Since this one is all random letters (instead of A, T, G, and C), we can assume pretty safely that this is an amino acid sequence.

Sidenote: I know I said it was all random letters, but all the letters actually represent certain amino acids. Just FYI.

Just for the sake of science, I’m going to use this as my example for the rest of the article.

Step 2: Choosing the Correct Search

At the BLAST homepage, you’ll notice that under “Basic Blast,” there are five different…. ummm… things.

Each of these things are types of BLAST search that you can run. I”ll try to write a post on the differences between them all, but for now, just know that we’re going to use “protein blast” because we have a protein query (a list of amino acids) and we want to find out what protein it probably is (by searching protein databases).

So, click on “protein blast” and we’ll move on.

Step 3: The Search Interface

If you’ve found this site (MPSN), then you’re probably VERY familiar with the one pictured below.

The screen that you use for the BLAST search isn’t EXACTLY the same, but there are enough similarities that we can compare them. In fact, the basic principles are ALL the same.

Do you see where it says “Enter accession number, gi, or FASTA sequence” ? This is where you’re going to paste the amino acid sequence.

In the entry field that says “Job Title,” you can come up with something to title your search if you’d like. But it won’t ruin your search if you leave it blank. I usually leave it blank.

“Query Subrange” and “Upload File” are other tools that may someday help you in a large BLAST search. But as for now, they’re pretty much useless to you.

Moving on down the page, we find that the next significant plaything is the database chooser-thingy. If you click on it, you’ll see a dropdown menu with a cornucopia of different databases you can choose. Don’t let this discourage you. The only one that’s going to help you most of the time is “Non-redundant protein sequences.” Although there isn’t a single database that holds all of the info at NIH, the non-redundant option holds “All non-redundant GenBank CDS translations + RefSeq Proteins + PDB + SwissProt + PIR + PRF.” If you don’t know exactly what that means, that’s ok. Just realize that it means that non-redundant is the choice you want to make.

If you’re dealing with a teacher who has told you what organism you’re studying, you can put the scientific name of the organism in the input marked “Organism Optional.” Although that may seem like common sense to you, I can’t tell you how many times I’ve seen people ask for help concerning that input.

ALMOST DONE HERE…

Now, all you need to do is make sure that the “blastp” is marked in the “Algorithm” box, and then you can click BLAST at the bottom of the screen. You should be redirected to a page that includes this:

Step 4: Interpreting Results

You did it! You conducted a BLAST search like a pro, but now what? Well, let’s start with that funky-lookin’ graph in the middle of the page.

This graph shows you different results that matched your search sequence. We gave the BLAST program a sequence and it compared our sequence to a few hundred thousand others (or more) and spit out the results that it found in the form of this graph.

You’ll notice that at the top, there are five colors: black, blue, green, pink, and red. There are also numbers that go with the colors. A common mistake is to think that these numbers represent the amount of amino acids that were matched in the result. For instance, if we had searched “FJLDKJ” and the BLAST had returned with a result of “FJDDKJ,” then that would give a score of 5 rather than 6. That’s not quite right…

The numbers you see are scores. They DO compare the amount that matched between the search and the result, but the don’t do that by counting the individual number. Think about it like a chapter exam in class: the number of questions isn’t always 100, but the score is always out of 100. This means that the teacher is “standardizing” the scores so that they all compare to each other on a scale of 100.

The BLAST system does basically the same thing, although the score is out of somewhere around 200. I’m not exactly sure what the maximum possible value is, but a 200 in blast is like an A on an exam. By that logic, I’m sure you can figure out that, in your blast searches, you want to use the results in the pink and the red.

Moving down the page again, you’ll see a section called “Descriptions.” This is the first real encounter with your results. What you see here is really where you can get a lot of information. For instance, I’ll bet that you didn’t know that the organism that our protein was made in is Saccharomyces Cerevisiae (baker’s yeast).

HOW DID I KNOW THAT? Just look at the results. The highest score AND the second highest score results both come from baker’s yeast. (You can click the little blue link on the left side in order to go to the pubMed article where the results are found. Clicking on that link sent me to an article that showed me not only the organism, but also that this protein is the result of the ARP2P gene.

OH! And I almost forgot to explain the E-Value!

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The E-Value is a fairly simple concept, once you get the hang of it. For those of you that are familiar with statistics, it’s really similar to the standard deviation. For the rest of you, allow me to explain: the E-Value is the probability (the percent chance) of there being a better result than this one. The smaller the E-Value, the smaller the chance of there being a better result. Therefore, a GOOD BLAST RESULT is one that has a HIGH SCORE and a LOW E-VALUE.

I really hope that this helps somebody out there.

Best of Luck,

Grey

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Do you remember that we decided that DNA and RNA are two separate languages? Transcription factors read “TATAAA” as “park here.” There are hundreds of other examples of how enzymes recognize only certain sequences, but we’re gonna keep it simple for now.

Well during translation, Dr. Ribosome and his tRNA graduate students “translate” the mRNA into yet ANOTHER language: polypeptide. Ok, after some thought, that’s too cheesy even for me. But since it gets the point across, I’ll leave it at that.

Once the mRNA strand has left the nucleus and entered the cytoplasm (or “cytosol” to some people), it is fair game for ribosome binding. That 5′ cap is EXTREMELY important here because it marks the location for the ribosome to bind.

WAIT! I want to add a little sidenote here that would have REALLY aided me in getting the concept of why all the little proteins are necessary. Remember how we needed transcription factors to start transcription? We’re gonna need translation initiation factors in order to start translation as well. But what’s really the point? You’ll have to read that article to find out, but for now, read on.

SO, the ribosome uses initiation factors to bind on the RNA, more factors find the AUG start codon, then the ribosome uses factors to bring the tRNA molecule with Methionine to the AUG Start codon… WHEN DOES THE RIBOSOME ACTUALLY DO SOME WORK BY ITSELF!?!?

Ummmmmmm, pretty much after that first methionine. You see, once everything is set up, the rest is automatic. If you look at the picture above, you’ll see that there is an “A Site,” a “P Site,” and a tRNA molecule floating away. The “A Site” is short of aminoacyl site, or “amino ACID” site. This is where tRNA molecules land if their anticodon matches the codon that is showing.The “P Site” is short for the peptidyl, or “PEPTIDEyl” site. This is where the amino acids bound to the tRNA molecules are added to the growing protein using a peptide bond. Finally, there is the exit site, where the empty tRNA molecule is allowed to float off and become recharged with another amino acid (of the same type).

In the case of the methionine start codon, the ribosome was moved so that the AUG codon was the only one that was showing in the Aminoacyl site. Once the methionine tRNA was bound, the RIBOSOME MOVED (not the tRNA molecule) and this pushed the tRNA molecule over to the Peptidyl site. This left an empty A-Site for another tRNA to enter and bind, causing the ribosome to move again. This time, the methionine tRNA is pushed into the exit site and ejected from the ribosome.

This move-eject-bind procedure will go on until the ribosome encounters a stop codon. If you look at the chart above, you’ll see three different combinations that will result in a stop. This happens because there’s a special molecule that possess and anticodon but NOT an amino acid. When this molecule moves into the P site, the ribosome will attempt to move the growing amino acid chain to bind with the new amino acid. The only problem is that with this special molecule, there is no amino acid. So the protein just floats off, and the ribosome falls apart, thereby ending translation.

Short Version

1. Factors bind ribosome to mRNA
2. Methionine tRNA initiates reading
3. Move – peptide bond/eject – Move – peptide bond/eject
4. Stop codon ends it all

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Best of Luck,

Grey

Have you ever baked cookies? Ok, so that’s a dumb question. But how about this one — did you use a cookbook? Unless you’re one of a seriously small percentage of humans with a photographic memory, then chances are that you did. So, making cookies was a 2 step process: writing the information down and deciphering that information into a different language.

I know it’s corny, but just go with it… for the sake of education.

From that awful analogy, have you figured out that there are 2 separate forces at work here? There’s the information-writing party and the information-reading party. In your case, the cookbook writers were… oh, you get it.

So, lets take what we now know and apply it to cell biology. In THAT case, the information-writing party would be anything involved in transcription, and the information-reading party would be anything involved in translation. Since this particular article is supposed to be about transcription, let’s go into a little more detail, but always keep in mind that everything in this process is about writing information down so that it can be read later.

Think of the genome as a library, full of cookbooks. This entire library is in the DNA language, and the ribosomes (the information-reading party) don’t speak DNA. There’s a wealth of information there for making all kinds of proteins, but the ribosomes just can’t read it, so it’s necessary that the information be translated into RNA, the ribosome’s native language.

The obvious place to start is in the nucleus at the gene of interest. We know from my post on the parts of a gene that there is a part called the promoter. I’ve heard the promoter referred to as everything from an on/off switch to an airport landing strip, and they all make sense. The promoter is the part of the gene that says “HEY! Bake THIS recipe!!”

There is a specific word in the DNA language that transcription factors, proteins that do speak DNA, understand as meaning “park here.” We refer to this word as a sequence called the TATA box. These transcription factors (there are many of them that work together to start transcription) act as a pit crew for RNA Polymerase. Their job is to find the starting line, get the RNA Pol in the right spot, and then give it any starting push that it may need (from stored energy, like ATP).

The RNA Polymerase is the only enzyme that is actually bilingual. Once it is given the proper push by the transcription factors, it glides along the DNA template strand, reading the DNA words and translating them into RNA. The two languages are very similar, in fact that only difference is that whereas the DNA alphabet is A, G, C, and T, the RNA alphabet is A, G, C, and U.

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At this point in the process, we have RNA sliding down the DNA template strand translating the directions from DNA into RNA. So, what next? There must be something programmed into the system to stop it at the end. Nope. In fact, transcripts don’t even make it to the end 100% of the time.

The RNA Polymerase glides along the top of the DNA strand in much the same way as a kid running down the curb on a street. It doesn’t take much force to knock the kid off the curb, and it takes even less to knock the RNA Pol off the DNA. There are no strong magnetic forces acting that hold the RNA in place, and the enzyme itself fits over the DNA like a horseshoe on a string. Sometimes, the transcript lasts all the way to the end of the coding sequence, sometimes it doesn’t, and sometimes it may go on for many thousands of base pairs after the gene has ended. Obviously, though, enough transcripts last until the end of the coding region, or else this system wouldn’t work.

You would think that, logically, as soon as the RNA Pol falls off the template strand, the new mRNA molecule would be finished and the process would be over. However, grammar is always important. I’m sure you can think of a few instances in which bad grammar made something hard to read, or even completely unreadable. Cells use a type of grammar as well, but we call it “post-transcriptional mRNA modification.” I know it’s a big scary term, but it simply means that after the RNA Pol does its job, the cell changes the molecule a little bit.

This modification consists of three very important parts: A 5′ cap, 3′ polyadenylation, and intron excision. These are all rather simple as well, but terms can be scary. When I say 5′ cap, I mean that the cell contains a host of enzymes that add a protective molecule onto the transcript at the 5′ end that signals the ribosomes to do their job later as well as protects the transcript from being sliced up by exonuclease enzymes. The 3′ polyadenylation, which means “lots of Adenosines on the 3′ end,” also protects the mRNA from exonuclease shredding. Intron excision, however, it probably the most important. In this process, chunks of DNA words that don’t actually mean anything are taken out of the directions in the mRNA.

Why these stinkin things are even there, we don’t know, but we DO know that if they aren’t taken out of the mRNA transcript, there will be no cookies later on. You see, in the DNA language, it is customary to take chunks of sentences and overlap them with each other. In order to read the original sentence, we must remove the chunks of other sentences. We call the original sentence chunks “exons” because we want to EXPRESS them, and the INTERVENING sentence chunks are called “introns.”

Once the cell has written down the recipe in a language that the ribosomes can understand and put it in the correct grammatical format, then the mRNA transcript can leave the nucleus and move on to the second step in protein expression.

The Short Version

1. DNA attacked by Transcription Factors that recognize the TATA box
2. RNA Polymerase binds to Transcription Factor complex, then begins to synthesize mRNA strand complementary to the DNA template
3. 5′ Capping, 3′ Poly-A tail, Intron Excision
4. Move out of Nucleus

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Best of Luck,

Grey

To do well with organic molecules, all you need to know is a few facts and to recognize some specific structures. That’s it. I rewrote this article three times, and every time was more detailed than the last. It eventually became ridiculous. I started by comparing a cell to a city, then to a house, then just explaining it in terms of how it relates to the body – UNNECESSARY. So, enjoy this; it’s short, sweet, and to the point.

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CARBOHYDRATES (sugars)

Ribose Monomer (5-Carbon Sugar)

Dimer of Glucose and Fructose

Cellulose (Glocose Polymer)

• Carbon, Hydrogen, and Oxygen. All sugars that you will deal with for a long time will be made up of a combination of these three elements. Many of them will be in a ring, like those pictured above.

D-Fructose

• Look for those elements in order to identify sugars. You can also look for a carbon backbone. Because carbon has 4 bonds, it can bond to another carbon on each side and still have room for binding to hydrogen or oxygen. Sometimes, the shape can give it away too, like glucose has six sides and ribose has five. But as you can see above, it isn’t necessary for sugars to be in a ring.

POLYPEPTIDES (proteins)

• ALL polypeptides are composed of amino acids, and ALL amino acids will have the following structure.

Amino Acid Structure by Tyagi Anuj

• There will be a Carboxyl Group, an Amino Group, a Hydrogen, and the dreaded “R-Group,” all bound to a central Carbon.

• Amino acids are the building blocks of proteins, in case you forgot. The “R-Group” is what makes one amino acid different from another; other than the R-Group, all amino acids are exactly the same.

• In order to identify a protein, look for a carbon bound to another carbon bound to a nitrogen, like in the schematic below. Although there are Hydrogens, Oxygens, and R-Groups bound to the middle ( you can ignore the arrows and dotted lines), you can see that there is a clear pattern of N-C-C-N-C-C-N-C-C-N… along the backbone of the protein.

Polypeptide Schematic by Dagmar and Ringe

• Your teacher may also hit you with a larger representation of a protein. Ribbons or blobs, like these pictured below, are usually proteins.

Src Protein

G Protein

LIPIDS

• Lipids have ONLY Hydrocarbons. Can you guess the elements that make those up? Carbon and Hydrogen? YES!

Octane

• That’s all you really need to know in order to recognize them. They are the only organic molecules that are made of just hydrocarbons.

Triglyceride

• You may need to know that squiggly lines like the ones pictured above are a shorthand way of writing carbon-carbon bonds. If you don’t see anything else written in on the squiggly line, you can assume that it’s just carbon and hydrogen.

NUCLEIC ACIDS

DNA Structure by Dr. Frank Boumfrey

• Three parts: Pentose (5-carbon sugar) + Nitrogenous Base (A,T,G,C) + Phosphate Group. These have a very distinctive shape, and you shouldn’t have any trouble recognizing them because they HAVE TO bond this way to be recognized as a nucleic acid. As you can see above, it’s got a structure that should be fairly easy to recognize.

• And don’t worry about the differences between different kinds of nucleic acids, the difference comes in the TYPE of nitrogenous base that is used. The overall structure of DNA, tRNA, mRNA, and whatever other -NA that you come across will all have the same basic structure… else they wouldn’t be a nucleic acid.

So Remember:

1. Protein: carbon bonded to nitrogen bonded to oxygen. and R group.
2. Sugar: carbon, hydrogen, oxygen
3. Lipids: carbon and hydrogen ONLY
4. Nucleic Acids: sugar, phosphate, nitrogenous base.

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Best of Luck,
Grey

EACH “LEVEL” OF PROTEIN STRUCTURE DESCRIBES SOME PART OF THE PROTEIN… and “PROTEIN”=”POLYPEPTIDE”… remember that. Keep them in your mind as you read the rest of the article.

A.) There are four levels of protein structure: Primary (first), Secondary (second), Tertiary (third), and Quaternary (fourth). Think about this like a stack of books: you can’t get to the third book without there being two books underneath, but you can have a stack of only two books without the need of a third or fourth.

Likewise, you can have primary structure without having secondary, or you can have tertiary structure without quaternary. If, at this point, you’re still unsure about exactly WHAT each of those are, it’s ok. Just make sure that you know that you must start at the bottom and work your way up and that every protein won’t have all four.

B.) The first level of protein structure DESCRIBES the order of amino acids in the protein. It has NOTHING to do with shape at this point. Eventually, these amino acids will effect the shape of the protein, but for now don’t worry about that.

When you think of Primary Structure, think back to kindergarten, when you were forced to stand in line between the same two people everywhere your class went. The primary structure of a protein is similar to the kindergarten line in that it focuses on the ORDER of the CERTAIN amino acids that make up that polypeptide. These lines of amino acids, bound to one another by peptide bonds, are the building blocks of the next level of structure…

C.) The second level of protein structure DESCRIBES the ways that the primary structure of the protein interacts with itself. This is the first instance of 3D shape that you see in polypeptides.

There are two major configurations that you need to know at this point, Aplha Helix and Beta Pleated Sheet. Actually, just to make things simpler, we’ll just call them helix and pleated sheet. The “alpha” and “beta” are just “the man” trying to keep you down. A helix is shaped like a tube and a pleated sheet is has ridges like a “Ruffle’s” potato chip.

In schematic representations of proteins, like these pictured here, flat ribbons are the accepted representation of beta sheets. Although they look flat, you’re just supposed to know that they are actually ridged.

fhuA transport protein

Bacterial Potassium Channel

In the examples given, the red alpha helices are shown in a bacterial potassium pump that is composed of only alpha helices, and the bets sheets are shown in the fhuA transport protein that is composed of only beta sheets. I’ll show in another post why these structures make them the best for their function.

Some amino acids will interact with each other to form shapes (helices or sheets). Remember concept A? It applies here. THERE IS NOTHING THAT SAYS THAT ALL AMINO ACIDS IN A PROTEIN HAVE TO FORM INTO HELICES OR SHEETS. Some just remain as strings of amino acids.

C.)  Tertiary structure is one of the easiest ones to remember. It DESCRIBES the way that the secondary structures interact… (there goes that “building blocks” thing again).

Blob. There you go. That’s tertiary structure. If you take your polypeptide “string” (primary), with all of its helix and pleated sheet “beads” (secondary), and you drop it on the floor, you’ll likely find that it falls into a bunch. Or maybe if you put the whole thing in your hand so that none of the strings hang off your palm, you’ll notice that you have to ball it up in order to get it all into your palm.

Tertiary structure is the EXACT SAME THING… kinda. it describes the way that the secondary structures interact with each other. Blob. Simple, huh? Theoretically, yes, but it’s at this point that proteins really begin to take on the shapes that they need to carry out functions in the cell.

Remember proteins are tools that the cell uses to do jobs. They aren’t the only tools, but they make up a whopping majority. The jobs that proteins can do depend COMPLETELY on their shape. You cant screw a screw with a hammer because it doesn’t have the correct shape.

D.) Finally… lets speed this up a little… Make four of those blobs from concept C and glue them together somehow. That’s quaternary structure. It describes the way that those tertiary structure blobs interact to form new and novel proteins that can do NEW JOBS. If you glue a hammer to a screwdriver… well, there’s probably not a lot that you can do with that, BUT THEORETICALLY, the possibilities are endless!

So, that’s it. Protein structure really gets more hype than it  deserves. But for your memory, here’s the condensed version:

• Primary – Kindergarten Line – certain arrangement of amino acids
• Secondary – Pringle’s tubes and Ruffle’s chips – alpha helices and beta pleated sheets
• Tertiary – Blob – interactions between primary and secondary structures
• Quaternary – BIG Blob – interactions between different polypeptide chains to form new shapes.

Here are some more links that may come in handy:

http://webhost.bridgew.edu/fgorga/proteins/proteins.htm#Primary

http://themedicalbiochemistrypage.org/protein-structure.html

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/D/DenaturingProtein.html

http://wiz2.pharm.wayne.edu/biochem/prot.html

Images courtesy of

Best of Luck,
Grey