Re: How Do restriction enzymes identify introns?

Mike,

I'm glad to help clear some things up for you.  It sounds like you're 
getting several different processes mixed up.  Prokaryotes (bacteria) 
typically do not have introns in their mRNA (though there are notable 
exceptions); eukaryotes (all higher organisms) almost always have introns 
in unprocessed mRNA.  Conversely, prokaryotes have restriction enzymes, 
while eukaryotic cells do not as such.  DNA cleavage and processing 
reactions in higher organisms typically are performed by more complicated 
systems as compared to the single protein restriction enzyme motif that has 
evolved in bacteria.

First, let me talk about restriction enzymes.  One way bacteria can evolve 
is by taking up DNA from other organisms; this is how resistance to 
antibiotics is transferred among various species.  On the other hand, there 
are numerous viruses that infect bacteria, inserting viral DNA in the 
bacterial cell.  It may be advantageous for bacteria to keep foreign DNA in 
some cases, and be able to get rid of it in others.  A related topic 
concerns repair of DNA damage, including mutations that are introduced 
during replication.

In both cases, bacteria must be able to differentiate between their own DNA 
and foreign DNA.  This typically is accomplished through methylation of the 
host DNA (though other modifications are possible).  During DNA 
methylation, an extra -CH3 group is added to DNA bases at somewhat 
irregular intervals by a methylation enzyme; sites of methylation are 
spaced distantly, rather than at every base or a single type of base (such 
as adenine).  The process of methylation occurs slowly, relative to the 
rate of DNA replication.  The methyl group serves as a tag so the cell can 
recognize which strand of DNA was the parent strand (the methylated one) 
from the daughter strand (non-methylated).  In the case of a replication 
error, the DNA damage control proteins know to change the base on the new 
strand of DNA as it has not yet been methylated.  Similarly, when foreign 
DNA invades a cell, it generally is not methylated.


This is where restriction enzymes go into action.  Each type II restriction 
enzyme has its own preferred recognition sequence, typically 4 to 6 base 
pairs, and typically palindromic (like AAGCTT or GCCG).  If they find their 
preferred sequence of DNA, and the DNA is not methylated on one of the 
strands, the restriction enzyme cuts the DNA on both strands.  These type 
II restriction enzymes are also called restriction endonucleases, since 
they can cut DNA anywhere the recognition sequence is found.  Once the DNA 
is cut, additional enzymes called restriction exonucleases come into 
action.  A second restriction enzyme called an "exo"nuclease binds to the 
ends of DNA (at a cut site, for example) and chews up the DNA as it moves 
in from the end.  So the endonuclease cuts the DNA, then the exonuclease 
chews it up, getting rid of the unwanted DNA sequence.



Intron removal is one example of the numerous types of RNA processing that 
occurs in eukaryotic cells.  All RNA is processed following transcription, 
including modifications to the 5' end of an RNA molecule, cleavage and 
acylation of tRNA, polyadenylation of mRNA, etc.  These typically occur in 
the nucleus prior to export of an RNA into the cytoplasm; if an RNA is not 
completely processed, or is processed incorrectly, they are degraded and 
recycled in the nucleus (another control mechanism ensuring only fully 
functional RNAs are used in their respective reactions).

RNA splicing occurs by several different mechanisms, depending on the type 
of processing that needs to be done; I'll just stick to messenger RNA for 
now.  As you know, proteins are synthesized by ribosomes using processed 
mRNA as the template.  However, the gene that encodes for a particular 
protein is actually composed of a series of exons (regions that code for 
the protein) and introns.  The introns are intervening sequences that must 
be removed before proteins can be made from the mature mRNA.  Though many 
theories are probable, nobody knows WHY introns are present in the gene of 
a protein (evolution is sometimes difficult to trace backwards); one theory 
is based on the fact that introns can carry signals for recombination 
reactions (where chunks of a gene from one chromosome are exchanged with 
the other).  In any case, the observation is that exons are roughly the 
same size (generally around 300 to 400 base pairs) and are interspersed by 
much larger introns (from 1000 base pairs up to tens of thousands).

The introns are removed by a spliceosome through a multi-step process.  The 
spliceosome is made up of several snRNPs ("snerps", small nuclear 
RibioNucleoParticles), the "U" class of snRNPs; each snRNP is comprised by 
snRNA (small nuclear RNA) and associated proteins (generally 7 to 14 
proteins per snRNA).  In the first stages of splicing, the U1 snRNP binds 
to the 5' splice site (on the 3' end of an exon, at the beginning of the 
intron); SF2 (splicing factor 2) and the U2 snRNP bind to the 3' splice 
site and the branch point (a short sequence near the 3' splice site).  The 
U1 snRNP and the U2 snRNP come together, bringing the two ends of the 
intron close together.  The U4/U6 snRNP complex (two snRNAs) and the U5 
snRNP bind; next, a conformational rearrangement occurs that results in 
release of U1 and U2 snRNPs, with the U4/U5/U6 snRNP complex bound to the 
splice sites.

In the first half of the reaction, the 5' splice site is cleaved and the 5' 
end of the intron is attached to the branch point making a lariat-shaped 
RNA.  Another conformational change occurs, then the free 3' end of the 
first exon is attached to the 5' end of the second exon.  The ligated exons 
are released, leaving the U4/U5/U6 snRNP bound to the lariat-shaped intron. 
 The two (transesterification) reactions are actually catalyzed by the 
snRNAs.  All the proteins associated with the snRNPs just seem to function 
to help the individual snRNPs find each other and stay in the correct 
functionally active conformations.
(I hope that wasn't too confusing.)

In the end, we have all of the introns removed and the mRNA.  Two other 
reactions occur before the mRNA is fully processed.  The 5' end has to be 
capped, and the 3' end is polyadenylated.  After these three reactions have 
occurred the mRNA is transported into the cytoplasm where it is transcribed 
by the ribosomes.


If you'd like some more reading on these subjects, or any other basic 
nucleic acid questions come up, I recommend reading "Genes" by Lewin.  It's 
a good collegiate level textbook that is fairly easy to read:

     Lewin, Benjamin
     "Genes VI"
     Publisher: Oxford; New York: Oxford University Press, 1997.
     Library of Congress number: QH430 .L487 1997


Regards,

Dr. Jim Kranz