Translation

Key Words


Ribosome: the machine that synthesizes protein by translating the code of the mRNA (with the aid of tRNA). It has a small and large subunit and is made mainly of RNA.
  1. tRNA: Transfer RNA is the adaptor molecule that ferries the amino acid to the ribosome. It has an anticodon; a three-base sequence that reads the codon.
  2. Codon: three-base sequence on the mRNA that encodes an amino acid
  3. Anticodon: three-base sequence on the tRNA that reads the codon. It is complimentary to it (mostly…there are some weird rules).
  4. “A-site”: Anterior (or "Amino-acyl) site on the ribosome. This is the site where the tRNA enters with the amino acid linked to it's 3' CCA sequence.
  5. “P-site”: Posterior site (or "peptidyl" site). This is the site in the ribosome where the tRNA with the growing protein chain is attached.
  6. “E-site”: exit site on the ribosome.
  7. Start Codon: the initiator tRNA, which holds the first amino acid reads the codon AUG and carries the amino acid Methionine (Met, or “M”). The anticodon for the start codon is 5'CAU (think about).
  8. Stop Codon: there are three codons that tell the ribosome to stop translation (UAG; UAA, UGA).
  9. Reading Frame: Since the code is read in groups of three, non-overlapping bases, Any stretch of mRNA has three possible reading frames. Only one reading frame at a time is used.
  10. ORF, or Open Reading Frame: A stretch of codons that starts with an AUG and ends with a stop codon and therefore can encode a protein. While I usually only write out a few, a typical ORF would encode hundreds of amino acids. Collagen, for example, is a large protein and is 1400 amino acids or so long.

Overview


I’m going to draw a bit on wikipedia for this. Here is a figure from them:
The quality of my reproduction apparently is poor. So, here's a link to the original.

Translation Overview
And here is a link to the video from HHMI. Translation in eukaryotes proceeds at about two amino acids per second. Bacteria is closer to 20 AA/second.

Regulatory sequences


Recall that there is a 5' untranslated sequence on the mRNA. There you will find sequences that direct the ribosome to the start of the protein-coding sequence. In bacteria, that sequence is more important and well characterized. In Eukaryotes, the regulation of where to start is less well understood…but we are learning.
This sets the reading frame:
Consider the sequence of letters BATHECATWASBADTHEDAYSHEBITTHEDOGOT, you can find the meaning only by starting at the correct letter (In this case, the third letter: THE CAT WAS BAD THE DAY SHE BIT THE DOG). If you start with BAT…that works…but the rest is not meaningful: HEC ATW ASB ADO).
The AUG tells the ribosome: put a Methionine here and keep reading in this frame. After some long series of amino acids, the Ribosome will encounter a stop codon, and release the mRNA (which does require a protein "release factor") and the newly made protein. Multiple ribosomes can be reading a single mRNA at one time, lined up one after the other.
The Code:
You need a triplet codon because you need three nucleotides to get enough possible combinations to encode all 20 Amino acids (there are 64 possible combinations, three of them are stop codons). The rest of the code is given below. Note that most amino acids have more than one codon. We say the code is “degenerate,” for this reason.
Here is the general code. Note that not every organism uses exactly this code. In a couple of organisms, UGA is read as a tryptophan codon, for example. How would this happen? Take a look at the where Tryptophan is in the codon table and predict what could change to lead to UGA becoming a tryptophan codon.

Code

More detail on the mechanism:


Of course, there is more…much more. Here is a really cool video (with an interesting sound track) from a lab that works on one of my favorite proteins: EF-Tu (elongation factor Tu is the name for the protein in bacteria. But, the function exists in eukaryotic systems also).
EF-Tu is arguably the original G-protein (certainly the first one we figured out). It sets the minimum time a tRNA must stay in the ribosome before the peptide bond is formed. In this way, accuracy is greatly improved.
Also, while there is some argument on the details, the GTP hydrolysis is thought to be part of what drives the assembly to "ratchet" to the next site.


The next video up should be this one:

it's good too.

RNA Splicing part 1


Take a look at
this video which is presented by Cold Spring Harbor Lab, where I used to work years ago and where some of the work I discussed today was done.
The parts of the mRNA that are removed and discarded are called "introns," while the parts that are maintained in the final, mature mRNA are called exons. The exons include the portion that codes for the protein, as well as other regulatory sequences both before and after the "coding" region. As the video describes, there are complexes of proteins and RNA that do the splicing. The whole complex is called the "Spliceosome." The components include the generically named "small nuclear RNAs" which assemble with proteins to form "
Small Nuclear Ribonuclear Proteins," or "snurps."
The story of how and why this happens is one of my favorite topics. We will discuss this more later.

RNA Processing


processing1All Most of what I’ll be talking about here occurs specifically in eukaryotes.
As usual, most images are from Wikicommons.
Following transcription, the “pre-mRNA” must be processed on its way out of the nucleus to the cytoplasm.
First, notice that the sequence that made up the promotor does not end up in the pre-mRNA. This is a general theme: With each step in the DNA→RNA→Protein pathway, the information needed to specify regulation of each step generally is lost as we go to the next step. This makes sense, since it is no longer needed. Jerry had the clever realization of that fact today in class.

Poly-A tail and GTP cap.


This addresses two issues: what is the "stop transcription signal" and how do we protect both ends of the mRNA from being rapidly broken down?
When the RNA polymerase reaches the end of the sequence that’s supposed to be transcribed, it hits a signal called the “polyadenylation signal.” This is the "stop transcription signal," but also results in something else happening. The sequence in the DNA (coding strand) is 5'-AATAAA. Of course, the polymerase is reading the template strand, so you could say the “signal” really is 3' TTTATT. The sequence varies a fair bit. This signals the RNA polymerase to leave the DNA. The sequence now in the pre mRNA (AAUAAA, or something similar) recruits a protein complex that will cleave the mRNA near the 3' end and an enzyme called PAP, for Poly Adenyl Polymerase, uses ATP to add a long series of A’s to the end of the mRNA. These A’s, and there may be hundreds of them, are NOT encoded anywhere. This is the reason there was a 3' extension at the end of the mRNA in the picture today. To be fair, the researchers already knew about this and it is part of how they oriented themselves on the DNA/RNA duplex).
The utility of the tail seems to be in recruiting several poly-A binding proteins that protect the mRNA from nucleases that degrade it, facilitate the next steps in processing, the transport of the mRNA out of the nucleus to the cytoplasm, and regulation of translation.
Some prokaryotes do a version of poly-A tail also. But, again, most of this applies to eukaryotes.
The other initial change is the addition of the GTP cap. Actually, it’s a modified G with a methyl group on position 7 of the base, cleverly called 7-methyl GTP. Wiki has a
short discussion of the mechanism of transfer. There is a specific enzyme that does the capping and the cap looks something like this:
GTPCap

Note the “inverted” 5'-5' link. The cap structure also protects the end of the mRNA from degradation.
RNA splicing:

The final processed mRNA might look something like this:

processed Transcript
It comprises the coding sequence, the cap, the 5' and 3' “untranslated region” (UTR) and the poly-A tail. I think all the names are pretty self-explanatory. The 5' UTR, in particular, will contain sequences that contribute to regulating translation. Only the green stretch above will make it into the protein.



RNA Splicing part more detail



Two benefits of splicing


While no one believes that RNA splicing evolved
because of these benefits, these are real benefits organisms now enjoy because of it.
  1. Alternative splicing: Not all the exons are included when splicing of specific mRNAs takes place. Different versions of the mRNA may be formed. In the example I gave earlier, exons 1, 3, and 4 could all be joined up in sequence, or, all 4 could be linked. This results in different versions of the protein, with different functions. The domain structure of proteins makes this possible. NOTE: you do not alter the order of the exons when this is done
  2. Exon shuffling: Also because of the domain structure of proteins, it is possible to add exons to genes via recombination and create different versions of the protein. Any chunk of DNA that has portions of introns on its ends that then is recombined (at the DNA level) into another intron will do no damage to an existing gene (It can be spliced out at the mRNA level). But, it also provides the possibility, through alternative splicing, to evolve proteins with new functions.
Key words:
Spliceosome: (there’s a good descriptive name) It’s the assembly of proteins and RNA that carry out splicing.
snRNPs (pronounced “snurps”). These are “small nuclear ribonuclear proteins.” They are the components of the spliceosome.
snRNAs: small nuclear RNAs are the main catalytic components. It is the RNA that carries out the reaction. They have names like “U1,” and “U2.” etc.


Self Splicing:
The story about how RNAs got spliced became more obvious when self-splicing RNAs were found. I mentioned these today.
The general reaction looks like this:
Splicing
There are two, successive trans esterification reactions that occur in a concerted way and use energy from an additional GTP that is brought in by the complex. The first takes a branch point sequence near the 3' end of the intron. The 2' OH of that branch point attacks the 5' exon/intron boundary. This splices the intron into a lariat and frees the 3' end of the exon, which then attack the 5' end of the next exon, resulting in the spliced exons and a lariat structure of the intron.
In the self-splicing form, “internal guide sequences” form base-paired structures that hold the players together and facilitate the attacks (which usually involve that 2' OH in an intermediate).
You can see below how the stem-loop structures formed using base pairs, then can fold into three-dimensional structures, facilitating catalysis. Yes, I know, it should have been obvious.

Self3

You can see how the base pairing can be used to bring the active sites together in more detail here. By the way, these last two images are used without permission. The one below is from the Molecule of the Month blog/discussion and the one above is from Trends in Ecology and Evolution.
Closeup

Normal mRNA splicing (non self-splicing)


The story is not all that different in the splicing of mRNA. There are guide sequences and a branch-point lariat. The chemistry varies a little as to who attacks whom when and where. But, the main difference is that the sequence of the intron is not that important for the structure to work. Instead of the intron folding into a complex structure, the separate RNAs in the snRNPs form those structures and then bind the specific sequences in the intron and at the intron-exon borders.




RNA Processing

Transcription`

Transcriptional regulation


As usual, images from wikicommons.
The first question is, how does the machine that makes RNA recognize a “gene?” In the past, I’ve called a “gene” the stretch of DNA encoding a protein
and its regulatory sequences. We can separate it into the “structural gene,” which contains the actual code of the protein as it would be found in the mRNA, and the regulatory sequences. A very basic regulatory region is called the “promotor.” It is a region that serves as the initial recognition site that says: “This is a gene, transcribe here.”
In addition to the hydrogen bonds that make up the “watson-crick” base pairs, there are additional sites for hydrogen bonds and other contacts that can be made without “unzipping” the DNA, primarily along the major groove. In particular, the major grove is just about the right size for an alpha helix of protein to fit in. There, R-chains on the outside of the helix can make specific contacts with the bases and “read” the sequence (that is, bind in a sequence-specific manner). The first step in transcription is recognition of the promotor, almost always upstream from the structural gene. There are several types of DNA binding proteins. Here are two examples:
LambdaRepThis is “Lambda repressor.” Notice two things: the protein binds as a dimer. In fact, each is identical and the overall DNA sequence is a “palindrome.” More on that later. Second, notice that one of the alpha-helices in each subunit is reaching down into the major groove, where it makes sequence specific contacts. This particular protein actually blocks access of the RNA polymerase in E. coli. But, similar proteins act to promote binding.
bZIPThis is a b-zip protein binding domain, of a class that acts to promote transcription in our cells. Notice the same two things. Even though the overall structure of the proteins is different (note that much of the structure is left out), the binding has some similarities.

Assembly of the transcription complex


What is the sequence?
Well, that depends. The promotor is the most important thing that determines what genes are expressed (turned on) in which cells. Thus, genes that have to be on in every cell have different promotors than those only on in some. Genes that are turned on following a signal cascade have specific promotors. One of the main “housekeeping” promotors has a sequence called the “TATA box” (I wonder if you can guess why. It is bound by a complex depicted below. This is known as a “General Transcription factor” because it is common to most genes.
As you can see, the
"Tata Binding Protein," or TBP interacts with the DNA over a larger region than just the TATA box. It also partially opens the DNA, where it bends it rather strongly. You may remember from the video we saw that there was a sharp bend to the DNA where the initiation complex was forming. It's also a little unusual in that it binds to the minor groove, using a beta-sheet domain. That bends and partially opens the DNA.
TBP
After initial proteins assemble at the binding site, they serve as binding sites for still more proteins that will be necessary to start transcription.

Here is a depiction of some of the proteins involved:
Transcription Factors

Notice the factors off to the right labeled 4 and 5. These have more to do with those larger levels of regulation I was talking about.

Here is a cool video on the process that shows some of the next step also:

The Operon

Operon:


After reading my blog, go to
this site and do the activity including the self test.
Also, here are two links to a site
about inducible promoters and another with a short list of available systems. Just scan them to get a sense of what is out there. In particular, you might want to read the short bit about the Lac promoter, towards the bottom of the chart on prokaryotic promoters.
Also, this is an interactive demo that is moderately useful. You have to put all the elements in place on the "DNA" to see it operating. It works if you have JAVA running on your computer. If you have it blocked, it will download and you can tell your computer to run it. It is up to you. You don't have to do this one.

An operon is a system that exists in bacteria, but not in eukaryotes, for regulating several genes together. Suppose, for example, you are a bacteria that sometimes encounters the sugar lactose. It would be good to have the genes for proteins to process that. You’d need a transport protein to get the lactose into the cell efficiently, and an enzyme to break the lactose into its components, Glucose (you know how that can be used) and Galactose (which you can also convert to glucose).
But, you wouldn’t want to be making these proteins all the time. It would be a waste of energy. Ideally, you would have a system that kept the genes for these proteins “Off,” but then be able to sense the presence of lactose and turn the genes “On.”
That is the
lac operon. And it serves as an example.

What an operon needs


  1. A stretch of DNA that encodes several proteins on ONE mRNA. The ribosome will make all of them reading the same message. This does not happen in Eukaryotes.
  2. Promoter: a site on the DNA recognized by RNA polymerase (which carries it’s own transcription factor to bind the promoter sequence, in E. coli).
  3. Operator: A sequence in the DNA that binds another protein, called Repressor. When repressor is bound, the polymerase cannot get access to the promoter.
  4. A Repressor: The protein that binds the Operator and prevents transcription. It is usually encoded nearby, transcribed from another promoter. Importantly, the repressor has to exist in two states: One that binds the operator and prevents transcription of the genes and one that does not.
  5. Effector molecule: molecule, such as lactose in the example of the lac operon, that binds to repressor and switches it between the two states.

Below is a general picture taken from Wikipedia.
The players are numbered below as:
  1. RNA Polymerase
  2. Repressor
  3. Promoter
  4. Operator
  5. Inducer (such as Lactose)
  6. 6, 7 and 8: the coding sequences for several proteins.
In the first panel, the repressor is bound because the inducer is absent. RNA polymerase cannot gain access to the gene.
In the second panel, the inducer changes the shape of the repressor, which causes it to release from the operator. Transcription can then occur.
Note, that there are also cases where the repressor binds only in the presence of the effector molecule. So, you can turn off an operon when the effector is present. This is great for feedback inhibition. The Trp operon is an example of this and is described in the book.
Operon