Week 15 Lecture 560B on Line

8/2/2019 Week 15 Lecture 560B on Line

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Lecture Week 15

Biochemistry of Nutrition, 560B

Dr. Charles Saladino

Introduction

For many years after the elucidation of the DNA structure by Watson and Crick, for which

they received the Nobel Prize, Central Dogma was the accepted theory. That theorybasically stated that DNA encodes for RNA, and then the RNA carries the DNA code in

such a manner as to code for the formation of a protein (now redefined as a polypeptide).

The synthesis of RNA from DNA is called transcription, whereas the protein synthesized

from the RNA code is called translation. For decades, this was always thought to be theorder of the sequence, until the mechanisms for retrovirus (viruses carrying only an RNA

code, instead of DNA) replication was elucidated. In this unusual case, the RNA actually

codes for DNA. Therefore, with the exception of the retroviruses using a reverse

transcriptase enzyme to allow RNA to code for DNA (as is the case with HIV), the CentralDogma is still correct for the encoding of DNA for specific polypeptides. In other words,

gene expression is the transformation of DNA information into functional molecules. It isthen that the DNA information becomes useful. This is a rich and complex subject,

requiring many chapters in a good biochemistry text. I will do my best to emphasize the

most important highlights, as we detail these mechanisms of transcription and translation inthis lecture.

Various RNA Types Described

Before describing the several types of RNA, let us examine characteristics common to allRNA species.1) RNA is a single stranded molecule. It is still composed of nucleotide units, but

there is no base pairing with a separate complementary strand.

2. The abbreviation RNA stands for ribonucleic acid, because the sugar is a ribose,

not the deoxyribose found in DNA. If you look at the deoxyribose sugar from last

week, you will see the lack of an OH group on carbon # 2, whereas the ribose sugar

does contain an OH group at that same position. Otherwise, the sugars (pentoses) arethe same.

3. Whereas DNA contains the G, C, A, and T heterocyclic nitrogen bases, RNAcontains G, C, A, and U (uracil see last weeks first figure). In other words, the

pyrimidine base, thymine, of DNA is replaced by uracil (U) in RNA, and thus U

is complementary to A, just the way G is complementary to C. Remember, bycomplementary we mean base to base pairing.

4. Except in the retroviruses, DNA is where the information of the genome is

archived, whereas the various RNA types are for transcription and translation.


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Messenger RNA (mRNA)

mRNA is formed from a single, genetically-active strand of DNA in complementaryfashion. The DNA strand that is coding for a mRNA is called the template strand, whereas

the opposite DNA strand that might not be active at that time is referred to as the coding

strand (even though it is not coding for anything at that point in time). Functionally,mRNA is the template for translation, but thats for later. For now, however, let us note

that a distinct mRNA is produced for each gene expressed in eukaryotes. Therefore,

mRNA is a heterogeneous class of biomolecules (500 6000 nucleotides).

Unique to most mRNA is a poly-A tail (about 200 adenine (A) nucleotides) found on the 3

end of the molecule. It is not transcribed from DNA. Rather it is added after mRNA is

transcribed by the enzyme polyadenylate polymerase. A consensus sequence (a series ofnucleotides that are involved in signaling and are not encoding genes) called the

polyadenylation signal sequence (AAUAAA) is found near the 3 end of the mRNA. It

signals the attachment of poly-A. It is known that these tails exist to help stabilize the

messenger and are also involved intransport of the molecule out of the nucleus and into thecytoplasm.

On the 5end is a CAP end consisting of a 7-methyl-guanosine attached backwards through

a triphosphate linkage, catalyzed by the nuclear enzyme guanylyltransferase. The addition

of the methyl group is catalyzed by guanine-7-methyltransferase. We will mention thefunctionality of these two end regions later. However, between these two ends is the

coding region of the mRNA.

mRNA is first formed as a larger precursor molecule, called hnRNA (heterogeneousnuclear RNA). So the portion of the mRNA that actually carries a genetic code derived

from DNA contains nucleotide sequences that are termed exons (one x, not like the Exxon

gasoline), plus non-coding introns. At first glance, it seems like a thermodynamic wastefor the DNA to code for parts of the messenger the can not translate a protein and thus have

no coding function. That would be the introns, as opposed to the exons which do carry a

useful genetic code from the DNA. So what is going on here? Well, as an mRNAmolecule matures, some of its sequences are removed, and these are known as introns.

What remains are exons (think of the x of exon to remember expressed). What splices

the introns out is the enzyme complex called a splicosome. Whereas a small number of

messengers contain no introns, most contain some, ranging in number up to around 50, asis in the case of the primary transcripts of collagen. Obviously, there must be consensus

sequences at each end of the intron that signal where the cut is to be made.

Splicing out introns allows rearrangement of the exon sequences, if so desired. This would

greatly increase the possibilities as to how a single gene could code for more than one

protein. For example, there would not be enough genetic material to code for all thedifferent antibodies that the body would require. By rearranging exons, different

transcripts could be derived from the same gene, instead of requiring one gene for every

possible antibody. So that is quite thermodynamically efficient after all!

As a point of interest, you might have heard the term snurps. These are small nuclear


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RNAs (snRNAs) that associate with protein (hence, small ribonucleoprotein particles =

snurps). They facilitate the splicing of exon segments by base pairing with the consensus

sequences at the end of each intron. I know you have all heard of systemic lupuserythematosus, an important autoimmune disorder that affects women in a ratio of about 10

to 1 over men. Anyway, the autoantibodies produced in lupus attack ones own proteins,

including the snRNAs.

Transfer RNA (tRNA)

This molecule folds upon itself and shows internal base pairing again, with itself, forming

a sort of clover leaf structure. Many of its bases become modified post-transcriptionally.

The tRNA is also made from a longer precursor, with an intron having to be removed fromthe anticodon loop (that term explained later) and from the 3 and 5 end of the molecule,

as shown in the figure below.

Other post-transcriptional modifications include adding a CCA sequence to the 3 terminalend, catalyzed by a nucleotidyltransferase, as well as modifying bases at various points

along the tRNA.

The purpose of this molecule is to carry an amino acid in its activated form to the ribosomefor peptide bond formation. There is at least one kind of tRNA for each of the twenty

amino acids. The tRNA consists of about 75 nucleotides (about 25 kd in mass), whichrenders it one of the smallest RNA molecules.

Ribosomal RNAs (rRNA)


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These are the major component of ribosomes. They serve a structural and a catalytic role

in protein synthesis (translation). Remember I mentioned in our enzyme lecture that

although almost all enzymes are proteins, some are not. This is because certain species ofrRNA have catalytic power (to be explained a little later). In eukaryotes rRNA synthesis

starts with a single precursor molecule referred to as preribosomal RNA and produces 5.8S,

18S, and 28S segments of rRNA. These S units are derived from the term Svedberg units,which is a relative measurements of a combination of molecular weight and shape, giving

rise to their sedimentation characteristics in a centrifugation process. Larger S values will

usually indicate larger molecular weight RNAs. The way these small S-value RNAs areformed is that the large precursor RNA is cleaved by ribonucleases to yield intermediates,

which are further trimmed to produce those rRNA species just mentioned. Also, some of

the proteins that are also part of the ribosomal structure will associate with the rRNA large

precursor before and during its post-transcriptional modification in the nucleolus of thenucleus.

This nucleoprotein will eventually be transported into the cytosol of the cytoplasm, where

the ribosome structure is assembled from its two main subunits. Whereas in bacteria, forexample, a 60S ribosome is assembled from one 30S and one 50S ribonucleoprotein

subunit, in eukaryotes, there are cytosolic 80S ribosomes, which are assembled from 40Sand 60S ribonucleoprotein subunits. By know you notice that numerical addition of S-

values is not valid. Again, that is because an S-value is a centrifugal sedimentation

characteristic based on both molecular weight and shape. You can visualize, I am sure,how two molecules of the same molecular weight but different shapes would sediment

differently.

RNA Polymerases

All cellular RNA is synthesized by a variety of RNA polymerases. Again, the synthesis ofRNA from a DNA template is called transcription. The requirements of the polymerases

are several. First, a template is required, usually double-stranded DNA, although both

complementary DNA strands do not have to be read at the same time. (RNA is not aneffective template, nor are DNA-RNA hydrid molecules.) Second, activated precursors are

required, meaning all four ribonucleoside triphosphates ATP, UTP, GTP, and CTP.

Third, divalent cations are required for the enzyme Mg2+ or Mn2+.

There are three unique RNA polymerases in the eukaryotic nucleus that transcribe the

various classes of RNA. These are large, multisubunit enzymes, with each type

recognizing specific types of genes. For example, RNA pol I synthesizes the large rRNAprecursor of which we have spoken. This occurs in the nucleolus in the nucleus, whereas

tRNA and mRNA are synthesized in the nucleoplasm. RNA pol II catalyzes the synthesisof the large mRNA precursor (hnRNA). Apparently some viruses use this pol to produce

viral RNA, and the enzyme can help synthesize some of the snRNAs. RNA pol III is

required for tRNA synthesis. Now lets see how this actually works, focusing on mRNAsynthesis. Please refer to the diagram below.


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So lets start at the very top of the figure showing the DNA template strand coding for itscomplmentary mRNA (hnRNA) strand. Note the DNA coding strand (which remember is

not coding for anything at the moment) is not present in the figure. Now if you look at the

mRNA, you will notice the 5 end on the left. This is because RNA pols synthesize new

RNA in their 5 to 3 direction. Ring a bell? You will also notice that the mRNAeventually will be read in triplets that is, sets of three nucleotides to designate one amino

acid. I will now insert below what we will call a codon table. Each triplet base sequence

in the mRNA is referred to as a codon.

To see how to use the table, you will notice the first column on the left for base # 1 of the

codon. Then there are four colums to choose from for the second base, and a final columnfor the third base of the mRNA codon. See if you can find how AUG designates the amino

acid methionine (Met) and CCA for proline (Pro). You will note that some amino acids

have more than one possible codon to designate them, which is why I mentioned that there

is at least one tRNA per amino acid this to be explain in a little while.

Print out this table (only) for the last exam, as you will need to refer to it during the

exam. The rest of the test is not open book.


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The rest of the figure preceding the above table is where we want to return to further

explain mRNA synthesis. The main part of the figure shows an enhanced view of the DNAtemplate strand that is coding for the mRNA. Notice the label flanking region on the 5side and a transcribed region to the right. Within the flanking region is a promoter region.

Please find it, and lets start there.

You will notice a CAAT box and a TATA box. These are consensus sequences, becausethey are signal, not bases from which genes are transcribed. Obviously, for RNA pol to

recognize specific genes from within huge stretches of DNA, it must know where the

transcriptional unit starts. This is why DNA templates contain regions called promotersites that specifically bind RNA pols, so as to determine where transcription begins.

Eukaryotic genes have promoter sites within a TATAAA consensus sequence called the

TAT box or Hogness box centered about -25 (negative, because it is about 25 nucleoridesfrom the beginning of the transcribing region). Many eukaryotic promoters also have a

CAAT box (GGNCAATCT) centered at about -75 nucleotides, as well as a GC box

(GGGCGG). Such boxes can vary from gene to gene. Also, gene transcription can be

further stimulated by enhancer DNA sequences, which can be kilobases away from eitherend of the transcribing region. The crude diagram below illustrates some of this.

In order to complete our explanation of the figure now above the codon table, you will

notice the transcribing region beginning in the vicinity of the CAP site. In eukaryotes,


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CAP structures are attached to the mRNA 5 end after transcription is complete, as are the

poly A tails. The figure also shows the DNA sequences for exons and introns, which we

described above briefly.

Finally, within the transcribing region of the DNA is a stop signal sequence to be coded

into the mRNA. However this is a stop signal for protein synthesis, which we have not yetdiscussed. Stop signals are mRNA codons and can be found in your codon table. Instead of

designating a particular amino acid, this codon sequence signals the end of a polypeptide

being formed. Codon UAA and UAG are codon stop signals.

Thus far, we have seen the rRNA synthesized in the nucleolus (using DNA as a template),

combined with protein, and transported to the cytosol as ribonuceloprotein, which forms an

assembly of ribosomal subunits and then a complete ribosome. The ribosome will be thesite of protein synthesis. In the mean time, genes are transcribed from the DNA code to

form a mRNA. It is first a big precursor, but after intron removal and exon assembly, the

mRNA is capped and a poly A tail added, which aids in the stabilization and transport of

the mRNA to the cytosol. This mRNA is carrying a DNA-directed genetic code (in theform of a series of usually contiguous, three-nucleotide codons) for the assembly of amino

acids into a very specific polypeptide.

tRNA as the Adaptor for Protein Synthesis

While all of this is going on, the tRNAs have already been synthesized from their specific

DNA segment, modified, and transported to the cytosol. There each amino acid to be

linked to its specific tRNA molecule is activated and then linked to its tRNA by an enzymecalled aminoacyl-tRNA synthetase, hooking the carboxyl end of the amino acid to the

tRNA. There is at least one specific aminoacyl synthetase and one tRNA for each amino

acid. If you think about it, by recognizing both the amino acid and the correct tRNA(because of the tRNA bases), this enzyme is really implementing the instructions of the

genetic code.

You will remember earlier in the lecture that I used the term anticodon (a three base

sequence) within the tRNA structure. It is near a partial loop in the tRNA opposite to the

end where the amino acid is attached. This is critical to understand: The anticodon will

match to the appropriate codon in the mRNA by complementary base pairing, rememberingthat there is no T in any RNA, and that U replaces T in its base pairing to A. Thus, every

properly made codon can accommodate an anticodon of a tRNA carrying an amino acid,

unless the codon is a signal. This is taking place upon the ribosome, where the mRNA hasbound. Polypeptide synthesis will be achieved when the amino acids of two adjacent

tRNAs connected to their respective codons form a peptide bond between the two amino

acids. The peptide bond is catalyzed by a peptidyltransferase. The enzymatic activity isintrinsic to one of the rRNAs (an example of a non-protein enzyme) within the ribosome.

The polypeptide will be synthesized in the amino to carboxyl direction, and the mRNA will

be translated in the 5 to 3 direction of the mRNA. What is important to note, is that the

codons of the mRNA recognize the anticodons of the tRNA and not the amino acids carried


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by the tRNA.

In order to keep adding amino acids, a translocase mechanism moves the ribosome threenucleotides toward the 3 end of the mRNA. This requires GTP as an energy source. This

continues until a stop signal is reached, and the polypeptide can fall off, become

associated with other polypeptides, and undergo a variety of chemical and structuralmodifications, including those we discussed at the beginning of the semester under the

topic of proteins. The actual synthesis of the peptide bond and movement of the mRNA

with the tRNA connected is difficult to explain here. Thus, I have included the followingdiagram to hopefully further clarify the process, trusting that it will not confuse you further.

Let me know if there are interpretation problems. OK?

Metabolism of Nucleotide Bases


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This subject is extremely complex and not necessary as a subject for an introductory course

in biochemistry. However, in keeping with the old saying, Sometimes a picture is worth a

thousand words, I have enclosed one more figure that gives you a feel for the synthesis ofpurines and pyrimidines. So I have two hopes, as I present this final figure, which I

admittedly scribbled out on a piece of paper. First, I want you to realize the important role

of amino acids and vitamins in the synthesis of these heterocyclic nitrogen bases, notmemorize every detail. Second, I hope you will not use this figure to do a handwriting

analysis on me!

Final Comments

There is obviously so much more to this story about the genetic code. Topics such as

jumping genes, genetic recombination, switching genes on and off, the telomere and

telomerase, oncogenes and cancer, retroviruses, along with many others are veryfascinating topics to explore. I hope sometime on your own that you do have the time to

look into some of these very interesting and challenging subjects. I tell you, the day will

come that coronary artery stents will be coated with not drugs, as that day is here already but with genes!

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Week 15 Lecture 560B on Line