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FUNdamentals
Chen
0918.08
11:00-12:00
Slide 1: intro slide
 1 hour on ribonucleic acids
 We will spend two hours on post-transcriptional regulation
Slide 2: Chapter 10
 Most material you can find from chapter 10; only look at the RNA part, not the DNA part
 You have already been taught about DNA; we will focus on RNA
Slide 3: Nucleotides and Nucleic Acids
 What are nucleotides and nucleic acids?
 Nucleotides and nucleic acids are biological molecules that contain a heterocyclic nitrogen base.
They contain nitrogen.
 What a nucleic acid is – it is a polymer of nucleotides, and they serve a central biological
purpose and that is transfer in the cell.
 Nucleic acids are important for information transfer in the cells
 There are two kinds of nucleic acids – one is DNA, one is RNA.
 RNA serves in the expression of genetic information stored in DNA through the process of
transcription and translation.
 However, RNA is there to express the genetic information stored in the DNA. Through the
process of transcription and translation – transcription is the process to make RNA. And then,
translation is the process you undertake - once you have RNA you translate RNA in a protein.
Slide 4: Information Transfer in Cells
 So here is just the flow of information transfer in the cells. Genetic information is encoded in
the DNA and transcribed via synthesis of RNA molecule.
 Genetic information is transcribed into RNA and the sequence of RNA is read by a ribosome.
 This RNA is read and translated into a sequence of amino acids. So that is a protein. DNA is
transcribed into RNA and translated in proteins. That is how the information is transferred in
the cells.
Slide 5: Figure 10.1
 This is just telling you how genetic information is transferred in a cell. Here is the double
stranded DNA; it can replicate by itself into 2 identical copies. One copy can transfer into
daughter cells.
 However, this genetic information needs to be translated into a protein which is here. The gene
here in the DNA needs to be translated into a protein. DNA cannot directly transfer into a
protein. They need to use a secondary or intermediate molecule which is RNA or mRNA which is
the subject of our lecture.
 So this genetic information needs to be transcribed into RNA. And then the RNA can be read
and translated into protein. That is how the genetic information is transferred from DNA to
protein inside the cells.
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Slide 6: What constitutes nucleotides
 Before we go into nucleic acids, I need to tell you what a nucleotide is and what constitutes a
nucleotide.
 If you take nucleotides and you complete lyse them, you will get 3 types of molecules. One is
nitrogen base, the second is a 5-carbon sugar and the third is phosphoric acid.
 They are in equal amounts, 1/3 nitrogen base, 1/3 sugar, 1/3 phosphoric acid.
 A nucleotide is composed of a nitrogen base, a ribose (5-carbon sugar) and a phosphoric acid.
Three parts in equal amount.
Slide 7: Nitrogenous Bases
 I am going to give you a few words about this – what is a nitrogenous base which constitutes a
nucleic acid. And, what are the 5-carbon sugars? The phosphoric acid is very easy; it is just a
phosphate.
 There are 2 kinds of nitrogen bases found in the nucleotide – one is pyrimidine and the second
in purine. You only find these in DNA and RNA. One is a purine and the other is pyrimidine.
 What is a pyrimidine? It is a six membered heterocyclic aromatic ring containing 2 nitrogens.
Remember is it a nitrogen base, so it contains nitrogen. It is a 6 carbon ring structure.
 There are 2 pyrimidines found in RNA – cytosine and uracil.
 What are purines? Purines, in contrast to pyrimidines, contain 2 rings. One ring resembles the
ring found on a pyrimidine, the other resembles an imidazole ring. They contain 2 rings.
 There are two purines that are found in DNA and RNA – adenine and guanine.
Slide 8: Figure 10.2
 So this slide shows the chemical structure of pyrimidines and purines.
 Pyrimidine is a 6 member ring; contains 6 atoms; 2 N’s.
 Purine contains 2 rings; one looks like a pyrimidine ring, the other resembles an imidazole.
When you see two rings, it is a purine. If only one ring, pyrimidine.
Slide 9: Figure 10.3
 And here is showing the structure of 3 pyrimidines. You don’t have to remember the chemical
structure – I won’t ask you that.
 You need know it looks like a pyrimidine. You don’t have to remember what cytosine looks like
– I won’t ask you how to draw the structure.
 There are 3 pyrimidines found in DNA and RNA. Cytosine and Uracil are found in RNA. Thymine
is only found in DNA instead of uracil.
 C and U are found in RNA.
Slide 10: Figure 10.4
 There are only 2 kinds of purines – one is adenine and the other is guanine. Both are found in
DNA and RNA.
 You don’t have to know the structure just the 2 purines that are found in DNA and in RNA.
Slide 11: The Sugar (ribose)
 The second part of the nucleotide is a sugar. What is a sugar? It is a pentose, or a 5-c sugar.
 It contains 5 carbons. And those found in the ribonucleosides contain D-ribose.
 Those found in deoxyribose lack oxygen. Only at the C2 position do they lack oxygen.
 The difference between DNA and RNA is that in DNA, there is no oxygen at the C2 position.
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So the ribose which constitutes deoxyribonucleoside is 2-deoxy-D-ribose.
However, the sugars found in DNA and RNA form a ring structure. They form a 5 membered
carbon ring structure known as furanose.
 You can call it D-ribofuranose in RNA – it is a 5 carbon ring structure.
 If it is a DNA, then we call it 2-deoxy-D-ribofuranose. It is a 2-deoxy-D-ribose from a 5 carbon
ring structure.
 And there are a number of atoms labeled 1’, 2’, 3’ – it is just to distinguish which is the number
of the nitrogen base. You go 1,2,3,4,5.
 Here the carbons are numbered 1’,2,3’ to distinguish the numbers of the base.
Slide 12: Figure 10.9
 Here is the structure of D-ribose here and D-2-deoxyribose. It is a 5 carbon sugar. Count them –
1,2,3,4,5.
 Then there is not oxygen found at the C2 position in the deoxyribose. Most sugars found in the
DNA form a ring structure here, like I just mentioned
 You only find this ribose from a 5 carbon ring structure. So you number it 1’, 2’, 3’, 4’.
 The only difference between DNA and RNA is at the C2 position you do not find oxygen in DNA;
you do find it in RNA.
 So that is the sugar. Sugar that forms a 5-carbon ring structure looks like this. The one found in
the RNA and the found in the DNA look like this. (Pointing to image.)
Slide 13: What are nucleosides?
 Now you have a base and a sugar – if you put these 2 together, you will form a nucleoside.
 What is a nucleoside? It is a nitrogen base plus a sugar that you call a nucleoside.
 The nitrogen base is linked to the sugar via a glycosidic bond.
 Then how do you name the nucleoside?
 Look at the example here. You add “idine” to the root name of a pyrimidine or add “osine” to
the root name of a purine.
 Here I give you an example.
o If the base is adenine, which is a purine linked to a sugar, ribose, now you call it
adenosine.
o When you see adenosine, you know it is a base linked to a sugar.
o Another example. When it says guanosine, you know it is guanine plus a sugar.
o Guanosine contains two kinds of molecules; one is a guanine, one is a sugar.
o Here is cytosine. It is a pyrimidine. When it is linked to a sugar, now you call it cytidine.
When you see cytidine, you know it is a cytosine linked to a sugar.
 That is how you name nitrogen bases to be nucleosides.
 You know that a nucleoside is a sugar plus a N base.
Slide 14: Figure 10.10
 This slide is showing you the structure of a pyrimidine. It contains only 1 ring. I know it is
pyrimidine linked to a ribose, so this is D-ribose through the glycosidic bond it is linked.
 This is purine is fused to a ribose through a glycosidic bond.
 This is the structure of a nucleoside. A base plus a sugar.
Slide 15: Figure 10.11
 So here is the multiring structure of cytidine. What is cytidine? It is a cytosine plus a sugar.
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Now here is cytosine plus a sugar. If it is adenosine, it is an adenine plus a sugar.
All these are ribonucleosides because it contains oxygen at the C2 position. It is not
deoxyribonucleoside. It is a ribonucleoside. It is a base linked to a ribose.
Here is another example. Guanosine means it is a guanine fused to a sugar.
That is the nucleoside.
Slide 16: What are nucleotides?
 If you have a N base and sugar, you call a nucleoside.
 What is a nucleotide? You have a nucleoside, then you have a nucleotide (plus a phosphate).
 This is the one found esterified to a cell.
 What are nucleotides - it is a nucleoside plus a phosphate.
 Nucleoside is a base plus sugar; nucleotide is nucleoside plus a phosphate group.
 Then this phosphate group is esterified to a sugar’s OH group; usually it is to a C5’ OH group.
 A ribonucleotide has a 5’ phosphate group.
 The number of phosphate at the C-5’ can be 1,2 or 3. You can contain 3 phosphate groups.
Either 1,2, or 3.
 I will show you the structure and how to name the nucleotides.
Slide 17: Figure 10.13
 What is this? Here is nucleoside (3AMP). (There was confusion in class about what he was
talking about – the caption clarifies the issue.)
 Here is adenine- so that is adenonsine. You can have 1 phosphate group fused to the C5’
position here. Then this is a nucleotide. Then you call this adenonsine 5’ monophosphate
because it only contains only phosphate group.
 adenonsine 5’ monophosphate. When you see this, you know it is adenine fused to a sugar, and
it only contains 1 phosphate group.
 Another example – uradine 5’ monophosphate. It is a uracil, fused to a sugar, containing one
phosphate. uradine 5’ monophosphate . Or you can call it UMP. It means that it is only mono,
P is phosphate, and the base is a uracil.
Slide 18: Figure 10.15
 Same thing; you can add 2 additional phosphates to AMP.
 For example, it is AMP because it contains one phosphate, but you add a second one to here
and call it ADP. The full name is adenosine 5’ diphosphate. That means you have 2 phosphates.
 If it contains 3 phosphates, then you call it ATP. Adenosine 5’ triphosphate.
 Then you name the phosphates by their adjecentness to the carbon – alpha, beta, gamma. The
one most adjacent to the carbon is alpha, then beta, then gamma. This is alpha phosphate,
gamma phosphate, and beta phosphate. You know then which phosphate is being referred to.
You don’t have to remember this, but now you know what is AMP, ADP and ATP and how many
phosphates are there.
Slide 19: Chart
 This is just a summary of what I just mentioned.
 There are 2 kinds of nitrogen bases – purine and pyrimidine. There are 2 purines found in DNA;
one is adenine, one is guanine.
 There are 2 pyrimidines. One is cytosine, the other is uracil.
 If you fuse a base to a sugar, it becomes a nucleoside.
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If you have adenine fused to a sugar – adenosine.
If it is cytosine fused to sugar, you called it cytidine.
And then you can fuse 1 or 2 or 3 phosphate groups to a nucleoside.
If you fuse 1 phosphate to adenosine, it is AMP. Fuse 3 phosphate, you call ATP.
That is easy to remember. I won’t ask you this anyway. But this is - what is a nucleoside and a
nucleotide? But you have to know what ATP is. You have to know that. You can’t just call it a
name; you have to have something in your mind – what is ATP?
Slide 20: What are nucleic acids?
 Okay now you have a nucleotide. Nucleotides form nucleic acids.
 Nucleic acids are polymers of nucleotides. You put each nucleotide linked together to get a
polymer. This polymer of nucleotides is a nucleic acid.
 The nucleotide is linked 3’-5’ by phosphodiester bridge.
 It is fused from 3’-5’. It is formed as 5’nucleoside monophosphate successively added to the
3’OH group of the preceding nucleotide. The preceding nucleotide contains the 3’OH group.
 Then the next nucleotide tide fused the 3’ OH group is fused to the 5’ monophosphate group.
 If it is a polymer of a ribonucleotide, it is RNA. The conventional way to read and write the
polynucleotide is 5’-3’ along the phosphodiester bridge. The phosphodiester bridge is link 3’-5’.
You read 5’-3’. You have to know you have read it from 5’ – 3’.
 If I don’t tell you that the sequence is AAGAC, what that means is it is 5’-3’ no matter what.
 Nobody will tell you the sequence is AAGAC and runs 3’-5’. It has to be 5’-3’. That is the way to
read the nucleotide sequence of a nucleic acid is from 5’-3’.
 The sequence actually passes from each phosphodiester bond from 3’-5’. It is opposite.
Slide 21: Figure 10.17 (this slide is really hard to follow; he continues to digress and repeat himself to try
to get across the point that linkage occurs 3’-5’ but read 5’-3’. Sorry for the impossible to follow
rendition of what he says.)
 I will show you this slide to avoid confusion.
 Here is RNA. The 5’ end is here. C5’. Here is the 3’ end. C5’ end.
 From here is 5’-3’. So what is the sequence of nucleotide? 5’-3’. ACGU. It is ACGU.
 So now it is read from 5’-3’. However, take a closer look at this phosphodiester bond. If you
read the sequence from 5’-3’ but the phosphodiester bridge passes from 3’-5’.
 5’ end of phosphate group fuses to the C3’ position. So the bridge you actually pass from 3’-5’.
You refer to 3’ of phosphodiester bond – what side are you referring to? (Points at board.)
 Here. Is it right? When you refer to the 3’ side of a phosphodiester bond, which side are you
referring to? Here? 3’ side of phosphodiester bond is which side? This side or this side. This
side!
 Phosphodiester bonds links from a 3’ carbon to a 5’ carbon on the next carbon.
 There are two phosphodiester bonds: (He also refers to these bonds as bridges.) A phosphodiester
bond is phosphate fused to oxygen. There are two phosphoester bonds. One is here (referring to
picture) and the other is here (referring to the picture).
 One of them is linked to 3’ position of the preceding nucleotide; the other one is fused to the 5’
position of the next nucleotide. When you read from the 3’ side of a phosphodiester bond, it’s this
side. (He keeps pointing to the picture).
 REMEMBER THERE ARE TWO PHOSPHOESTER BONDS. ONE IS FUSED TO THE 3’ CARBON THE OTHER
IS FUSED TO THE 5’.
 This is important because the nucleus can cleave either side of a phosphodiester bond.
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The nucleus can cleave here or here (referring to the picture). Where you cleave will give you a
different product.
You must know what product will be generated; this depends on where cleavage occurs.
You read a sequence from 5’ to 3’. In the slide, you read the sequence as A-C-G-U.
It’s NOT U-G-C-A; this is wrong.
The phosphodiester bonds are opposite from the way you read the sequence (aka 3’ to 5’)
Slide 22: TGCAT
 This next slide shows a simplified version of the nucleic acid sequence.
 To be simplified, the vertical line is the sugar.
 The diagonal slash is to show the phosphodiester bond.
 The identity of nitrogen base is up at the top.
 The sequence is T-G-C-A-T.
 The picture on the right side of the slide is even more simplified than the image on the left side of
the side because the labels of 5’ and 3’ have been removed.
 When you mean T, it means thymine + sugar + phosphate.
 Even more simplified, you can just write T. Anybody who understands this will understand what the
structure will look like.
Slide 23: What are the Different Classes of Nucleic Acids?
 There are two main classes of nucleic acids found inside cells.
 One of them is DNA- deoxyribonucleic acid.
 The other one is RNA- ribonucleic acid.
 There is only ONE kind of DNA.
 DNA serves one purpose only- to store genetic information.
 There are four types of RNA. There are four different purposes.
 One is messenger RNA, the 2nd one is ribosomal RNA, and the 3rd one is transfer RNA. We will focus
on these three.
 In addition to these RNA types, you can also find small nuclear RNA and small non-coding RNA.
 Small nuclear RNA is important for mRNA splicing- will learn what splicing is tomorrow.
 Small non-coding RNA is important for post transcriptional gene silencing- will not talk about today.
Slide 24: Principal Kinds of RNA Found in an E. coli Cell
 There are three types of RNA in E. coli- mRNA, rRNA, and tRNA.
 This chart shows you the relative size of the RNA.
 Only pay attention to the last column, the percentage of Total Cell RNA.
 mRNA only constitutes 2% of the total RNA. If you isolate the total RNA from the cells, only 2% will
be mRNA.
 You will find that 80% of the RNA is ribosomal RNA.
 What is mRNA mRNA encodes proteins; it’s very important but only constitutes 2%.
 Why is rRNA so abundant? When you translate mRNA, you need ribosomes. rRNA constitutes the
ribosome. One mRNA can be read with many ribosomes. That is why you need more rRNA. You
not only make one copy of mRNA inside of cells, you can make tens of thousands of copies inside
the cell of the same mRNA. Then you have to translate it at the same time. That’s why you need
more ribosomal RNA.
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Ribosomal RNA is important, but mRNA is more important because it is the mRNA that encodes
proteins.
Without mRNA, you will not be able to make proteins.
The cell cannot function without proteins, but mRNA only constitutes 2% of the cell.
Slide25: Messenger RNA
 What is messenger RNA?  it carries the genetic information that is stored in DNA and translates
the genetic information into proteins.
 mRNA will translate into proteins.
 The mRNA found in prokaryotic and eukaryotic cells are quite different.
 In prokaryotic cells, you only have one single mRNA that can synthesize more than one protein. One
mRNA can translate into several different proteins.
 In eukaryotic mRNA, a single mRNA codes for one protein. That’s the difference between
prokaryotic and eukaryotic mRNA  a single eukaryotic mRNA codes for a single protein; a single
prokaryotic mRNA codes for the synthesis of many proteins.
Slide26: Eukaryotic mRNA
 Tomorrow, we will get a slide to show that one mRNA only makes only one protein in eukaryotic
cells.
 There is another difference between eukaryotic RNA and prokaryotic RNA.
 In eukaryotic cells, DNA is transcribed to produce heterogeneous nuclear RNA.
 What is heterogeneous nuclear RNA?  it’s a sequence containing introns and exons with a poly-A
tail. This will be mentioned more tomorrow.
 Eukaryotic mRNA has heterogeneous nuclear RNA containing introns and exons.
 An intron is an intervening sequence and an exon can encode proteins.
 An intron cannot encode protein; an exon can encode protein.
 mRNA should translate into a protein, but the mRNA is synthesized as pre-cursor mRNA which
contains introns.
 Introns need to be removed in order to translate into a protein because introns cannot translate
into a protein.
 It also contains a poly-A tail. A reaction called “splicing” will produce the final mRNA without the
introns.
 Introns found in the precursor mRNA need to be spliced out in order for this heterogeneous nuclear
RNA to become a mature RNA so it can be translated.
 The process to remove introns is splicing.
Slide 27: Ribosomal RNA
 What is ribosomal RNA?
 A Ribosome is composed of two components.
 One component is RNA and other component is protein.
 A ribosome contains RNA and a protein.
 2/3rd of ribosome is rRNA; 1/3 of the ribosome is protein.
 rRNA is a scaffold for ribosomal proteins; it is important for assembling ribosomes.
 rRNA forms complicated secondary structures.
 The relative size of rRNA is referred to as “S” or sedimentation coefficient.
 It is just the measurement of the relative size of the rRNA.
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Slide28: Complicated 2ndary of rRNA
 This is just an image of the very complicated, 2ndary structure of ribosomal RNA.
Slide 29 Ribosomes:
 This slide shows the difference between a prokaryotic ribosome and eukaryotic ribosome.
 Won’t go into much detail, but for the lecture in translation, you should be taught more about the
ribosomes.
 For this lecture, you will not need to specifically need to know the specific sizes of the RNA.
 There are two subunits no matter if you are referring to prokaryotic or eukaryotic subunits.
 Ribosomes contain two subunits—a small subunit and a large subunit. You refer to the size as “S”.
For example, in prokaryotes you have 30S (the smaller subunit) and 50S (the larger subunit).
 The 30S subunit contains 21 proteins (this is the small subunit) and contains one type of RNA, a 16S
RNA.
 The larger subunit, which is 50S, contains 31 proteins but comes in two kinds of RNA—23S and 5S.
 You don’t have to remember, but you have to remember for this lecture that there are two
subunits that contain RNA and protein in both prokaryotes and eukaryotes.
 Subunits are similar between the prokaryotic and the eukaryotic cell. The sizes are different though.
 rRNA is important in the assembly of the ribosome which is responsible for the translation of mRNA.
Slide 30: Transfer RNA
 In order to translate mRNA you not only require rRNA, you also require tRNA.
 mRNA cannot be translated by itself; you require rRNA and also tRNA.
 What is tRNA  it is a very small polynucleotide; it usually contains 73-94 residues.
 Each tRNA is less than 90 nucleotides long.
 This tRNA also folds into a complicated secondary structure.
 Each amino acid has at least one unique tRNA.
 What’s the function of tRNA?  it carries amino acids to the ribosome.
 Each amino acid has at least one specific tRNA.
 For example, methionine. There is one tRNA specific for methionine. Sometimes there can be two
or three tRNA for a single amino acid.
 For example, cysteine is an amino acid that can have three different tRNAs capable of carrying it to
the ribosome.
 Each amino acid has at least one tRNA to carry the amino acid to the ribosome.
 The 3’ terminal sequence of a tRNA is usually CCA; the “A” is the most terminal one and fuses to the
amino acid.
 The tRNA fused with an amino acid is called aminoacyl tRNA; this means the tRNA has been charged
with an amino acid; this is a substrate for protein synthesis.
 Amino acid + tRNA = aminoacyl tRNA.
Slide 31: Secondary structure of tRNA.
 Here is the secondary structure of tRNA.
 The 3’ end is a single strand and will be charged with amino acids.
Slide 32: DNA & RNA Difference?
 We now know the structure of RNA and we learned the structure of DNA earlier this week.
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What is major difference between the DNA and the RNA?  The major difference is that in DNA,
you cannot have oxygen at the C-2’ position.
RNA contains an –OH group at the C-2’ position; because RNA contain vincinal –OH groups at the C2’ and C-3’ position, this makes the RNA very susceptible to hydrolysis.
Because DNA does not contain a C-2’ –OH group, it is more stable.
DNA is more stable than RNA because it does NOT contain an –OH group at the C-2’ position.
This makes sense because DNA needs to be very stable because genetic information is stored in
DNA; if DNA was not stable you would get mutations like cancer and die.
Genetic information is transcribed to RNA and RNA makes protein.
When you don’t need the protein, what can you do?  You can degrade the RNA or stop its
synthesis.
You can also degrade RNA that has been synthesized; this is why RNA should be very unstable. You
must degrade RNA when you don’t use it, but you can always synthesize the RNA again.
You don’t want to degrade your DNA; you want the DNA to be stable.
That’s the difference between DNA and RNA.
Slide 33: Hydrolysis of Nucleic Acids
 Here are a few words about RNA and DNA.
 RNA is resistant to dilute acids.
 RNA is hydrolyzed by a diluted base.
 If you treat with RNA with dilute base, you hydrolyze the RNA.
 But BOTH DNA and RNA are hydrolyzed by nucleases.
 Nucleases are enzymes to degrade RNA and DNA.
 Remember this RNA is resistant to dilute acid, but RNA is very susceptible to dilute base. Both
DNA and RNA can be hydrolyzed by enzymes called nucleases.
Slide 34: SKIP
 Skip this slide; will not ask questions about this.
Slide 35: Nucleases
 Here are a few words about nucleases.
 Nucleases are enzymes which hydrolyze DNA or RNA.
 Nucleases, generally speaking, are phosphodisesterases.
 What is a phosphodiesterase?  an enzyme that cleaves phosphodiester bonds.
 This is why nucleases are also called phosphodiesterases because they cleave phosphodiester
bonds.
 The cleavage can occur at EITHER side of the phosphate group---either at the 3’ side or the 5’ side.
 There are two kinds of nucleases.
 With one, you can hydrolyze DNA internally. This is anywhere inside and it is called an
ENDONUCLEASE.
 There is another type of nuclease that cleaves from the terminal side. It can cleave from the 5’ side
or the 3’ side. These are called EXONUCLEASES.
 Endonucleases cleave internally and exonucleases cleave terminally either from the 5’ or 3’ end.
 Also, it can be cleaved from the 3’ end which is the “A site.”
 It can also cleave at the “B site” which is the 5’ side.
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Know that there are two enzymes that cleave phosphodiester bonds- endonucleases and
exonucleases.
Slide 36: Figure 10.30
 If the exonuclease cleaves at the 3’ side, it is CALLED “A” CLEAVAGE.
 If it is cleaved at the 5’ side, it is called “B” CLEAVAGE.
 If the enzyme cleaves at the “A” bond, you get the product 5’ nucleoside monophosphate. This is
the product you get when you cleave at the A site.
 If the enzyme cleaves at the “B” bond, you get 3’ monophosphate products.
 Microphone goes out at 49.34.
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