Week 14 Lecture 560B on Line

8/2/2019 Week 14 Lecture 560B on Line

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13Week 14 LectureBiochemistry of Nutrition, 560B

Dr. Charles Saladino

Introduction

In spite of great differences in lifestyle and appearance, organisms tendto exhibit striking similarities at the molecular level. We have seen thatthe metabolic activities of basically all cells rely upon common groups ofmolecules that include amino acids, carbohydrates, and lipids, often inpolymer form. Each molecular type has a signature of physiologicalfunction, chemical composition, and interactions with otherbiomolecules. For example, the DNA is a repository for our geneticinformation. We shall start by describing its structure.

Nucleotides

I am sure you know that DNA (deoxyribonucleic acid) and RNA(ribonucleic acid) are what are commonly referred to as nucleic acids.Most biochemistry texts describe the metabolism, structure and functionof nucleic acids and their components reasonably well. Unfortunately,your text frankly trivializes the subject of nucleic acids by its glaringomissions. With that said, we can start by noting that both of thesebiomolecules (DNA and RNA) are long polymers that are able to carryinformation in a manner that can be passed from one generation to thenext, whether it is at the level of the organism, the cell, or the molecular

level. Both forms of nucleic acids consist of a very large number offundamental units, known as nucleotides. Each nucleotide is composedof a sugar, a phosphate, and a heterocyclic nitrogen base. The commonbackbone of DNA and RNA is the sugars linked by phosphate groups.The bases vary, as will be explained in more detail later, and they areinvolved in nearly every aspect of cellular life. To be a little specific,nucleotides participate in oxidation-reduction reactions, transignalingsequences, energy transformation, and biosynthesis reactions.Remarkably, nucleotides also play structural and catalytic roles in thecell. In fact, I will stick my neck out a bit and state that no other class ofmolecules participates in such varied functions and those essential to

life as do the nucleotides.

Nucleotides show structural diversity, and they are quite ubiquitous.They are also capable of having more than one phosphate attached, asin ADP and GDP or ATP and GTP, for example. I have included in a figurebelow the structures of the five (by far) most prominent bases that helpmake up nucleic acids. Notice that two, adenine (A) and guanine (G) arereferred to as purines, being double ring structure. On the other hand,


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thymine (T), cytosine (c), and uracil (U) are pyrimidines and are singlering structures. Lets note right here that DNA contains A, T, C,and G, whereas the various RNAs contain A, U, C. and G. Inother words, the T of DNA is replaced by the U in all RNA molecules, and,thus, we normally do not find U in DNA or T in RNA. As you probably

know, fluorouracil is an analogue of thymine, and is used inchemotherapy. This is because it gets substituted into the DNA ofcancer cells (and unfortunately many normal cells) and interferes withnormal DNA synthesis. This then blocks cell replication, although notwithout severe, toxic side effects.

SugarsThe basis upon which the names of the two classes of nucleicacids are based appear to rest with the sugar. Thus, deoxyribonucleicacid contains the sugar deoxyribose in the backbone, whereas thebackbone of RNA contains the sugar ribose. Below is a figure showingthe structure of the deoxyribose, a five carbon sugar manufactured

(remember?) in the pentose phosphate pathway. Please note that it istermed deoxyribose, because there is no OH group at carbon numbertwo. On the other hand, the ribose sugar does contain an OH group inthe carbon number two position.Now lets link the nucleotide together in a single strand.This shows apolynucleotide segment linking by phosphodiester bonds. Notice thatalthough there is one A, T, C, and G base, respectfully, linked to adeoxyribose sugar, there can be any arrangement of bases, for ex,TTTAGCCGT. We shall see later on that the order of the base sequenceis what ultimately determines the genetic code. Please take note ofwhat might seem trivial, but what turns out to be a very important

detail. That is, notice that on one end of the polynucleotide is a 5phosphate attached to the OH OH group on carbon # 5, whereas on theother end is a 3 hydroxyl group. This is almost always the case. The symbol behind the 5 of the phosphate and the 3 of the hydroxyl istermed prime. Thus, we have a 3 prime hydroxyl and a 5 primephosphate end, neither of which is linked to another phosphate, andthey represent their respective ends of the polynucleotide chain. Thiswill become very important for DNA synthesis. We should note here thatone characteristic of naturally-occurring DNA molecules is their length.

As you probably know, the DNA structure was elucidated by Watson and

Crick (for which they received a Nobel Prize). They utilized x-raydiffraction crystallography, and it was in the 1950s before the computerwould have been available for such work. As I am sure you also know,the DNA structure is a double -helix, wherein two polynucleotide strands are wound around one another in a right-handed twist. Toachieve this, we require a second polynucleotide strand, just like thatone above. (Devlin or any on-line source can easily be found to observethis double helix.) So what holds the two polynucleotide strands


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together? The short answer is hydrogen bonds. The longer answercomes now. Lets look at the figure below.It not a coincidence that inthe figure above, A is shown paired to T with a double hydrogen bond;and G is shown paired to C with a triple hydrogen bond. This is the basepairing arrangement under normal conditions: A-T and G-C. Notice one

pyrimidine is paired to one purine, because the spacing arrangementwould not be stable with two purines or two pyrimidines paired up toeach other, respectively.You will notice that it is the base pairingthrough hydrogen bonding that holds the two polynucleotide strands ofthe helix together. The fact that G pairs to C and T pairs to A is referredto as complementary base pairing, and that represents the only normalbase pairing combination. To the left of the base pairing figure is the -helix. The term refers to the fact that the helical twist is right handed ,with the polynucleotide chains coiled around a common axis. The sugar-phosphate backbones occupy the outside position, with the purine andpyrimidine bases lying on the inside of the helix. The bases are nearly

perpendicular to the helical axis, and there are ten bases (on each side)per turn (360 degrees) of the helix. The double helix is stabilized by Vander Waals forces between adjacent bases on the same strand and by thehydrophobic effect - mentioned to you when I described the entropychanges in the water shell near a protein.

A striking feature of DNA is its length. Even in the simplest organisms,such as the polyoma virus (which can cause cancer in some organisms),the DNA is 5100 nucleotides in length. This corresponds to 5100 x 2 =10, 200 bits or 1275 bytes (1 byte = 8 bits. Now the E. coli genome,which is a single DNA molecule comprising two chains, is 4.6 million

nucleotides (1.15 megabites of information).

The human genome is approximately 3 billion nucleotides dividedamong the various chromosomes. Just for your interest, there is anAsiatic deer with a genome about as large as that of humans. However,it has only three sets chromosomes, with the largest chromosomecontaining more than 1 billion nucleotides! If this chromosome were tobe stretched out, it would reach one foot in length. Amazing how allthat is packed into a cell nucleus. Believe it or not, some plants containeven larger DNA molecules. Finally, please note the term kb (kilobases).So for example, if we say that a particular mammalian chromosome

contains 250,000 kilo base pairs. That means 250,000 x 1000 basepairs.

Now please notice one important feature in the above diagram of thebase pairing. One polynucleotide strand runs in the 5 to 3 direction (onthe left), whereas the complementary strand (on the right) runs 3 to 5.This arrangement of the polynucleotide strands is referred to asantiparallel. Please remember this important feature.


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We should note that bacteria contain a single helix, but it is in a closedcircle. The same applies to mitochondrial DNA. Yes, mitochondriacontain DNA, but not enough to encode for all the mitochondrialproteins. Thus, by way of example, some of the electron transport

system proteins are coded for by mitochondrial DNA, whereas others areencoded by the nuclear DNA.

Reversing Melting of DNA

As we shall discuss shortly, during replication of DNA and duringtranscription, the two strands of the DNA double helix must beseparated from one another. at least in one local region of the longDNA. In the laboratory, where I once studied DNA repair, I usedto disrupt the DNA helix by heating it in solution. Heating will disrupt

the hydrogen bonds between the base pairs, allowing thepolynucleotides to separate. One can also do this in a very alkalinesolution, which ionizes the base pairs and disrupts the base pairing.This disruption of the helix is sometimes referred to as melting. The Tmis defined as the melting temperature. Because the double bonds of A-Trequire less energy to break than the triple bond of G-C, DNA with ahigher G-C content than that of A-T will show a higher Tm value.However, once the strands separate, these complementary nucleotidestrands can spontaneously reform a double helix. This renaturationprocess is often called reannealing. Although this sounds like test tubework, during DNA replication and during transcription of RNA, this

reassociation of the two strands is crucial for the biological functions ofDNA. However, inside cells, helicase enzymes are present to facilitatethis ATP-requiring denaturation during DNA synthesis.

Finally, DNA from two different species can be denatured and permittedto reanneal or hydridize, in the presence of each other. The hybrid DNAduplexes (with each organism contributing one DNA strand of the doublehelix) will form; and the degree of hybridization will be an indication ofhow closely related the genomes of the two organisms are. Further,similar hybridization experiments with DNA and RNA can locate genes incellular DNA that correspond to a particular RNA (to be discussed in our

next lecture).

DNA Synthesis

It is intuitively obvious that without a mechanism for maintaining thefidelity of the genetic code from one generation to the next, and that


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means at the cellular level too, the survival of the next cell generation,and thus the species, would never be possible. The mechanism bywhich DNA is replicated produces two identical and complete DNAmolecules from an original DNA whose code is determined by the orderof occurrence of each of the four types of nucleotide bases. With only

four different bases, but with literally millions of each one occurring invarious combinations and permutations, probably about 100,000 genesmight be present in the genome to code for an even larger number ofpolypeptides (that later point to be explain in the next lecture). Thus,our genetic code simply is which of the four bases occurs in what orderand how many of each there are. Remember, eventually we shall seethat one gene codes for one polypeptide (not one protein, again, nextweek). This week, our task is to explain DNA synthesis (replication).Then we will be in a good position discuss the characteristics of thevarious RNA species next week.

The molecular mechanism of DNA synthesis involves more than 20proteins that interplay with one another. Among the most prominentare the DNA polymerases (properly abbreviated DNA pol - singular),which promote the formation of bonds joining new nucleotides together.Specifically, DNA pol catalyzes the step by step addition of nucleotidestogether, with the new DNA strand being assembled directly on apreexisting template which will be complementary to the new growingpolynucleotide. Observe the figure below.

This figure starts with DNA synthesis already in progress. Well will go

back to the initiation of it later. Please notice several things. First,notice that the growing strand is antiparallel to the template. That is,the growing strand is running in the 5 to 3 direction, whereas itstemplate is running 3 5. The template represents one of the twooriginal DNA strands of the double helix, after the helix has beenunwound in this area of the whole DNA by a helicase enzyme. Only oneof the two original DNA strands is shown in this figure; and it is thestrand on the right that starts with an AAC sequence. At some point,the opposite strand of the original DNA will act as a template for a newpolynucleotide strand in the same manner.

It is important to note that the DNA must unwind, in order to engage inreplication. It does not open up like a zipper from one end to the other.Can you imagine the logistical nightmare of doing that???? It is far toolong (much longer than the cell) for that to happen. Thus, it opens uplocally at what is called a replication fork. This is what happens. Agenetic signal occurs wherein an endoculease enzyme puts a break inthe sugar phosphate backbone, so that the helicase can unwind thestrands. The strands now separate, so that both of the original DNA


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strands can act as a template, wherein proper base pairing can occur, asis shown in the figure above. Note that a G is being added, because thetemplate is a C. Note also that what is being added is a dGTP. The d isfor the deoxyribose sugar of the nucleotide, the G is obviously thepurine guanine, and there are three phosphates in this nucleotide. As

the nucleitode is being added by the polymerase, the two endphosphates are cleaved off, so that a nucleotide is added (one sugar,one phosphate, and one base. The removal of the two phosphates (PPi)which are subsequently split into two single phosphates (Pi + Pi) arewhat provide the energy for this endothermic, endergonic reaction ofDNA synthesis. We should note that the replication of DNA requires DNApol that takes it instructions from the DNA template.

The synthesis of DNA thus produces two new DNA strands from oneoriginal DNA. This is called semi-conservative DNA synthesis,because each new DNA is half new and half old (the latter half from the

original DNA molecule). If you understood the above material, then youwill notice that new growing strands of DNA always synthesize in the 5to 3 direction, antiparallel to their respective templates. I will nowplace a diagram for you to observe, and I ask you to pay particularattention to what is labeled the leading strand and then the laggingstrand.

You will notice that because the leading strand runs 5 to 3, it can justkeep growing until the synthesis is halted in that area of the replication

fork. However, the opposite growing strand, called the lagging strandmust also grow in its 5to 3 direction. That means that new pieces ofgrowing DNA must grow from behind the previous growing strand i.e.back fill. Thus you can deduce from this, as well as by rechecking thefigure above the present figure, that the polymerase requires a free3OH group to which to add the next nucleotide. That is why one newstrand of DNA is made continuously, whereas the other new strand issynthesized in fragments.

DNA Synthesis Requires an RNA Primer

Having just stated that the DNA pol that adds a nucleotide to a growingstrand requires a free 3OH group, we can ask how the process of DNAsynthesis can start at a new replication fork when there is nothing toadd to in the first place. The answer is that an RNA primer must besynthesized on the template to provide that free 3OH group to initiateDNA synthesis. That RNA primer is synthesized by a special RNA polcalled primase. This then synthesizes a short stretch of RNA (maybe 5


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nucleotides in length) that is complementary to the DNA templatestrand. This seems counterproductive, as the primer will have to beremoved later; but the answer lies in the fact that the DNA pol can notstart polynucleotides de novo, whereas RNA pols can.

You will notice in the above figure the term Okazaki fragments. Theseare the segments (about 200 nucleotides long) of newly growing DNAthat result from back filling as lagging strand segments. Each wouldhave to begin with an RNA primer.

Now obviously, these primers do not remain as part of the newlygrowing DNA strand. Thus they must all be removed; and indeed theyare by an exonuclease enzyme. This enzyme sequentially chops out theprimer. Now a different DNA pol will fill in the gap left by the RNAprimer, and all thats left to do is to seal the bond. That last step iscarried out with an enzyme called DNA polynucleotide ligase, which

does require ATP as an energy source. Thus, DNA synthesis in general isenergy intensive. A figure showing the ligase action is presented below.

Replication Rate and Replicons

DNA replication is substantially slower in eukaryotes than in prokaryotes(like bacteria). Specifically, in a eukaryotic cell, about 50 nucleotidesper second are added to a growing DNA strand one tenth the rate ofthat of prokaryotes. This difference is undoubtedly due to the complex

structure of the chromosomal material found in eukaryotes. For thelast exam, look up the structure of chromatin.

In spite of this relatively slow rate of DNA synthesis, eukaryotic DNAsynthesis can be accomplished in a matter of hours in a given cell. Thatis quite a feat, considering the very large amount of DNA in the humangenome, because if we take the above-described eukaryotic nucleotideaddition rate of about 50 per second, it would take about a month toreplicate the chromosomal DNA (about 150 million base pairs) in a givencell! So how does the cell get around this problem? Again, the design isso intelligent. The answer is multiple replicons (areas around a

replication fork origins of replication) operating simultaneously tocompress the replication time of their large genomes. In humans areyou ready for this replication requires about 30,000 replicons, witheach chromosome containing hundreds!

Now think about what such a system requires. How about (for onething) a mechanism that each sequence is replicated once and onlyonce? In the cell cycle, DNS synthesis and cell division are coordinated


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so as to ensure that the synthesis of all DNA sequences is completebefore the cell can progress into the next stage of the cell cycle. Thisrequires several checkpoints that control the progression through thecell cycle.

Finally, I can not cover all the proteins involved in DNA synthesis in thislecture. It has gotten long enough already. However, to further try andstimulate great awe for the way the cells biochemistry works, I willclose with this the following:

Besides the various proteins we have already discussed (DNA pols,replicase, helicase), once the strands of DNA are separated by thehelicase, they must be stabilized by the binding ofreplication proteinA. This is a single-stranded DNA binding protein. Then DNA synthesisbegins with the binding of DNA pol , which is the init iator pol. Now,hear this. This enzyme has primase activity, as well as DNA polymerase

activity. Make sense? After about 20 deoxynucleotides have beenadded to the primer, another replication protein (protein replicationfactor C PFC) displaces DNA pol and attracts another proteinassociated with replication, called proliferating cell nuclear antigen(PCNA). The PCNA then binds to DNA pol . This binding probably causes an allosteric change in the DNA pol that cause this enzyme to be extremely active and quite suitable for synthesizing long stretches ofnew DNA.

What I have described here is called polymerase switching. This isbecause the pol has replaced the pol . Finally, and again amazingly,

pol has 3 to 5 (yes thats different tha n that we have discussedbefore) exonuclease activity. This exonculease activity can then act asa proofreader and edit the replication of DNA (meaning removemismatched bases thus minimizing mutations). Finally, as we havealready discussed, replication continues in both directions from theorigin of the replication fork, until adjacent replicons meet and ligate.While all this is going on, the RNA primers are being removed, aspreviously described.

Arent you just at least a little impressed that all this works as well as itdoes? I am.

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Week 14 Lecture 560B on Line