dr okunowo wahab's introductory molecular biology lecture note ii

8/14/2019 Dr Okunowo Wahab's Introductory Molecular Biology Lecture note II

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INTRODUCTORY

MOLECULAR BIOLOGY

Okunowo O Wahab, Ph.D.

Biochemistry Department

College of Medicine

University of Lagos


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Course outline

STRUCTURE COMPOSITION OF DNA&RNA

TYPES OF DNA

TYPES OF RNA

DNA REPLICATION

POST-REPLICATIVE MODIFICATION OF DNA

CHEMOTHERAPIES TARGETING REPLICATION

RNA TRANSCRIPTION

POST-TRANSCRIPTIONAL MODIFICATION OF RNA

GENETIC CODE

TRANSLATION.


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DNA structure

Composition of DNA,deoxyribonucleic acid, andRNA, ribonucleic acid.

In slide 1, the DNA molecule isseen as a double stranded helix

or a ladder shaped molecule.

The rungs of the ladder areactually hydrogen bonds (H) joining two nucleotide basestogether.

The sides of the ladder madeup deoxyribose sugars are joined together byphosphodiester bonds.

Slide 1


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In slide 2, a nucleotide is seen to becomposed of 3 chemical groups: anitrogenous base or nitrogen containingbase, a sugar molecule & a phosphate (PO4)group.

There are 2 types of nitrogenous basesfound in DNA or RNA known as purines orpyrimidines; a nucleotide contains eitherone.

There are 5 purine or pyrimidine bases foundin DNA or RNA; these are adenine, thymine,guanine, cytosine, or uracil.

This nitrogen containing base is linked to a 5carbon sugar either deoxyribose, whichhas a hydroxyl group (OH) at carbon 3,

or ribose, which has OH groups at carbons 2& 3.

The phosphate group is attached to thesugar at carbon 5.

Note that each nucleotide has a direction the top part is the 5-phosphate end &the bottom is the 3-OH end

Composition of

DNA&RNA


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Each nucleotide can be linked togetherendlessly to form a nucleic acid. Thephosphate group of the lower nucleotidelinks to the OH group at the 3 carbon on

the upper nucleotide to form aphosphodiester bond.

The 5 end will always will have a free orunbound phosphate group & the 3 endwill always have an unbound 3-OHgroup. Direction is an importantconcept to remember!

Each side of the DNA helix or ladder is inthe opposite direction of each other.

This is called an anti-parallel structure.Each side is always anti-parallel.

Also note that the ladder structure isfurther stabilized by hydrogen bonds that join the nitrogenous bases together,which as are the rungs of the ladder

Slide 3

Slide 3


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The H bonds are very systematic in how they form. Ofthe 4 bases in DNA - adenine, guanine, cytosine &thymine. Adenine always bind to Thymine & Guaninealways bonds with Cytosine.

These are called base pairs or Watson-Crick base

pairing after the two scientists who proposed thestructure of DNA in the early 1950s. This is also calledcomplementary base pairing bases only bond withtheir complement.

In RNA, 3 of the 4 bases are the same as in DNA adenine, guanine, cytosine -- the only difference is uracil

is present in place of thymine. Thus, base pairs in RNAare Adenine & Uracil and Guanine & Cytosine.Complementary base pairing is a very importantconcept to keep in mind
http://www.accessexcellence.org/AB/BC/James_Dewey_Watson.htmlhttp://www.accessexcellence.org/AB/BC/James_Dewey_Watson.html


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On the right side is asegment of DNA, whereH bonds form between A-T & C-G and nucleotidesare linked together in ananti-parallel fashion.

Therefore, DNA is acomplementary & anti-parallel structure.

On the left side of theslide, RNA tends to be

single-stranded, but it canloop on itself and when itdoes, it is also in acomplementary & anti-parallel fashion.

Slide 4


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Types of DNA

The double helix of DNA hasbeen shown to exist in severaldifferent forms, depending uponsequence content and ionicconditions of crystal preparation.

The B-form of DNA prevails underphysiological conditions of low

ionic strength and a high degreeof hydration.

Regions of the helix that are richin pCpG dinucleotides can exist ina novel left-handed helicalconformation termed Z-DNA.

This conformation results from a180 degree change in theorientation of the bases relative tothat of the more common A- andB-DNA


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Characteristics of Types of DNAParameters A Form B Form Z-Form

Direction of helical rotation Right Right Left

Residues per turn of helix 11 10 12 base pairs

Rotation of helix per residue(in degrees)

33 36 -30

Base tilt relative to helix axis(in degrees) 20 6 7

Major groove narrow and deep wide and deep Flat

Minor groove wide and shallow narrow and deep narrow and deep

Orientation of N-glycosidicBond

Anti Anti Anti for Py, Syn for Pu

Comments most prevalent within cells occurs in stretches ofalternating purine-pyrimidine

base pairs


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Types of RNA

RNA is similar to DNA. Like DNA, it is composed of nucleotides joined by 3- to 5-phosphodiester bonds, the purine bases adenine and guanine, and the pyrimidinebase cytosine. However, its other pyrimidine base is uracil rather than thymine.

In RNA, the sugar is ribose, which contains a hydroxyl group on the 2-carbon.

RNA chains are usually single-stranded and lack the continuous helical structureof double-stranded DNA. However, RNA still has considerable secondary andtertiary structure because base pairs can form in regions where the strand loops

back on itself.

As in DNA, pairing between the bases is complementary and antiparallel. But inRNA, adenine pairs with uracil rather than thymine. Basepairing in RNA can beextensive, and the irregular looped structures generated are important for thebinding of molecules, such as enzymes, that interact with specific regions of theRNA.

The three major types of RNA (mRNA, rRNA, and tRNA) participate directly inthe process of protein synthesis. Other less abundant RNAs are involved inreplication


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Messenger RNA (mRNA)

It comprises only about 5% of the RNA in thecell yet is by far the most heterogenous typeof RNA in terms of size.

The mRNA carries the genetic informationfrom the DNA to the cytosol, where it is usedas the template for protein synthesis.

Each mRNA molecule contains a nucleotidesequence that is converted into the amino

acid sequence of a polypeptide chain in theprocess of translation

Special structural characteristics of eukaryoticmRNA include a long sequence of adeninenucleotides (a poly-A tail) on the 3-end ofthe RNA chain, plus a cap on the 5-end

consisting of a molecule of 7-methylguanosine attached backwardthrough a triphosphate linkage.


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Ribosomal RNA (rRNA)

Ribosomes are subcellular ribonucleoprotein

complexes on which protein synthesis occurs.

Different types of ribosomes are found inprokaryotes and in the cytoplasm andmitochondria of eukaryotic cells.

rRNA is found in association with a number ofdifferent proteins as components of the

ribosomes.

There are three distinct size species of rRNA(23S, 16S, and 5S) in prokaryotic cells andeukaryotic mitochondria.

In the eukaryotic cytosol, there are four rRNAsize species (28S, 18S, 5.8S, and 5S).

rRNA helps to hold mRNA on the ribosome inorder that mRNA can be translated.

{Note: S is the Svedberg unit, which is relatedto the molecular weight of the compound.}Together, they make up 80% of the RNA in thecell.


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Transfer RNAs (tRNA)

Transfer RNAs (tRNA), the smallest ofthe three major species of RNA molecules(4S), have between 74 and 95 nucleotideresidues.

There is at least one specific type of tRNAmolecule for each of the 20 amino acids

commonly found in proteins.

Together they make up about 15% of theRNA in the cell.

Each tRNA serves as an adaptormolecule that carries its specific amino

acid to the site of protein synthesis. There,it recognizes the genetic code word thatspecifies the addition of its amino acid tothe growing peptide chain.

Typical clover leaf structure of

tRNA


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THE CHEMICAL NATURE OF RNA DIFFERS

FROM THAT OF DNA

RNA is a polymer of purine and pyrimidine ribonucleotides linked together by 3,5-phosphodiester bridges analogous to those in DNA. Although sharing many features withDNA, RNA possesses several specific differences:

(1)In RNA, the sugar moiety to which the phosphates and purine and pyrimidine bases areattached is ribose rather than the 2-deoxyribose of DNA.

(2) The pyrimidine components of RNA differ from those of DNA. Although RNA contains theribonucleotides of adenine, guanine, and cytosine, it does not possess thymine. Instead ofthymine, RNA contains the ribonucleotide of uracil.

(3) RNA exists as a single strand, whereas DNA exists as a double-stranded helical molecule.However, given the proper complementary base sequence with opposite polarity, the singlestrand of RNA is capable of folding back on itself like a hairpin and thus acquiringdoublestranded characteristics.

(4) Since the RNA molecule is a single strand complementary to only one of the two strands ofa gene, its guanine content does not necessarily equal its cytosine content, nor does itsadenine content necessarily equal its uracil content.

(5) RNA can be hydrolyzed by alkali to 2,3 cyclic diesters of the mononucleotides, compoundsthat cannot be formed from alkali-treated DNA because of the absence of a 2-hydroxylgroup. The alkali lability of RNA is useful both diagnostically and analytically


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Summary

DNA is a double stranded helix rungs joined together by H bonds phospodiester linkages between 5 & 3 ends of nucleotides; 5 3 direction contains deoxyribose sugar contains A, T, C, G nucleotides

found in the nucleus of cells; some is found in the mitochondria

RNA is primarily a single stranded molecule linked by the same type of phosphodiester bonds that join DNA together; 5 3

direction contains ribose sugar

contains A, U, C, G nucleotides 3 types of RNA messenger, ribosomal & transfer (mRNA, rRNA, tRNA) found in the cytoplasm; however it is manufactured in the nucleus, so some is found

there


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DNA Replication The genetic information stored in the nucleotide sequence of DNA serves

two purposes. (1) It is the source of information for the synthesis of all protein molecules of

the cell and organism

(2) It also provides the information inherited by daughter cells or offspring.

Both of these functions require that the DNA molecule serve as a templatein the first case for the transcription of the information into RNA

and in the second case for the replication of the information into daughterDNA molecules.

Replication of DNA occurs during the process of normal cell division cycles


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The mechanics of DNA replication was characterized in E. coli.

3 distinct enzymes capable of catalyzing the replication of DNA were identifiedas DNA polymerase (pol) I, II, and III.

Pol I is most abundant but has as its primary role to ensure the fidelity ofreplication through the repair of damaged and mismatched DNA.

Replication of the E. coligenome is the job of pol III which is much lessabundant than pol I, however, its activity is nearly 100 times that of pol I.

There have been 5 distinct eukaryotic DNA polymerases identified, , , , and .

The ability of DNA polymerases to replicate DNA requires a number ofadditional accessory proteins such as:

1. Primase

2. Processivity accessory proteins (dnaA binding protein)3. Single strand binding proteins (SSB)

4. Helicase

5. DNA ligase

6. Topoisomerases

7. Uracil-DNA N-glycosylase


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Steps in DNA Replication


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Steps involved in DNA replication. The figure above describes DNAreplication in an E coli cell, but the general steps are similar ineukaryotes.

A specific interaction of a protein (the O protein) to the origin ofreplication (ori) results in local unwinding of DNA at an adjacentA+T-rich region.

The DNA in this area is maintained in the single-strand conformation

(ssDNA) by single-strand-binding proteins (SSBs). This allows avariety of proteins, including helicase, primase, and DNApolymerase, to bind and to initiate DNA synthesis.

The replication fork proceeds as DNA synthesis occurs continuously(long arrow) on the leading strand and discontinuously (short

arrows) on the lagging strand.

The nascent DNA is always synthesized in the 5 to 3 direction, asDNA polymerases can add a nucleotide only to the 3 end of a DNAstrand.


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DNA Replication is semiconservative

DNA replication is semiconservative. During a

round of replication, each of the two strands

of DNA is used as a template for synthesis ofa new, complementary strand.

The double-stranded structure of DNA and the template

function of each old strand (dark shading) on which a

new (light shading) complementary strand issynthesized.


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Post-Replicative Modification of

DNA: MethylationOne of the major post-replicative reactions that modifies the DNA is methylation.

The sites of natural methylation (i.e. not chemically induced) of eukaryotic DNAis always on cytosine residues that are present in CpG dinucleotides.

However, it should be noted that not all CpG dinucleotides are methylated at the

C residue.

The cytidine is methylated at the 5 position of the pyrimidine ring generating 5-methylcytidine.

Methylation of DNA in prokaryotic cells also occur to prevent degradation of host

DNA from its restriction endonucleases.

This also helps prokaryotic cells to degrade invading viral DNAs. Since the viralDNAs are not modified by methylation they are degraded by the host restrictionenzymes.


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Chemotherapies Targeting Replication

Alkylating Agents

The class of compounds that have been used the longest as anticancerdrugs are the alkylating agents which includes cyclophosphamide (Cytoxan,Neosar), ifosphamide, decarbazine, chlorambucil (Leukeran) andprocarbazine (Matulane, Natulan).

React with and disrupt the structure of DNA (DNA fragmentation).

Some catalzye the cross-linking of bases in the DNA which prevents theseparation of the two strands during DNA replication.

Some agents induce mis-pairing of nucleotides resulting in permanent

mutations in the DNA.

Alkylating agents act upon DNA at all stages of the cell cycle, thus they arepotent anticancer drugs. However, because of their potency, prolonged useof alkylating agents can lead to secondary cancers, particularly leukemias.


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Topoisomerases Inhibitors

Several classes of anticancer drugs function through interference withthe actions of the topoisomerases.

Two of these classes are the anthracyclines and the camptothecins.

The anthracyclins inhibit the actions of topoisomerase II whose functionis to introduce double-strand breaks in DNA during the process of

replication as a means to relive torsional stresses.

Etoposide (VP-16, Vepesid, Etophos, Eposin) functions throughinhibition of topoisomerase II

Camptothecins inhibit the action of topoisomerase I, an enzyme that

induces single-strand breaks in DNA during replication

Anthracyclines also function by inducing the formation of oxygen freeradicals that cause DNA strand breaks resulting in inhibition ofreplication.


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Antimetabolites In order for DNA replication to proceed, proliferating cells require a pool of

nucleotides.

The class of anticancer drugs that has been developed to interfere withaspects of nucleotide metabolism is known as the antimetabolites.

There are two major types of antimetabolites used in the treatment of abroad range of cancers

componds that inhibit thymidylate synthase and compounds that inhibitdihydrofolate reductase (DHFR).

Both of these enzymes are involved in thymidine nucleotide biosynthesis.

Drugs that inhibit thymidylate synthase include 5-fluorouracil (5-FU, Adrucil,

Efudex) and 5-fluorodeoxyuridine.

Those that inhibit DHFR are analogs of the vitamin folic acid and includemethotrexate (Trexall, Rheumatrex) and trimethoprim (Proloprim, Trimpex).


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RNA transcriptionBacterial RNA transcription is described in

four steps:

(1) Template binding: RNA polymerase(RNAP) binds to DNA and locates apromoter (P) melts the two DNA strandsto form a preinitiation complex (PIC).

(2) Chain initiation: RNAP holoenzyme(core + one of multiple sigma factors)catalyzes the coupling of the first base

(usually ATP or GTP) to a secondribonucleoside triphosphate to form adinucleotide.

(3) Chain elongation: Successive residuesare added to the 3-OH terminus of thenascent RNA molecule.

(4) Chain termination and release: Thecompleted RNA chain and RNAP arereleased from the template. The RNAPholoenzyme re-forms, finds a promoter,and the cycle is repeated. Thetermination is brought about either by theaction of Rho-protein or hairpin loopformation of the RNA transcript


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The relationship between the sequences of an RNA transcript and its gene, in whichthe coding and template strands are shown with their polarities.

The RNA transcript with a 5 to 3 polarity is complementary to the template strandwith its 3 to 5 polarity.

Note that the sequence in the RNA transcript and its polarity is the same as that inthe coding strand, except that the U of the transcript replaces the T of the gene.


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Post transcriptional modification of RNA

When transcription of bacterial rRNAs and tRNAs iscompleted they are immediately ready for use intranslation. No additional processing takes place.

Translation of bacterial mRNAs can begin even beforetranscription is completed due to the lack of the nuclear-cytoplasmic separation that exists in eukaryotes

In contrast to bacterial transcripts, eukaryotic RNAs (all 3classes) undergo significant post-transcriptionalprocessing.


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Removal of Introns from RNA

All 3 classes of RNA aretranscribed from genes thatcontain introns.

The sequences encoded bythe intronic DNA must beremoved from the primarytranscript prior to the RNAsbeing biologically active.

The process of intron removalis called RNA splicing.

Introns are removed bynucleophilic attack orsnNRP(small nuclearribonucleoprotein)


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5'-Cap of Eukaryotic mRNAs

The addition of guanosine triphosphate is catalyzed by

Guanyltransferase.

Methylation is catalyzed by guanine-7-methyl transferase.

Eukaryotic mRNAs lacking the cap are not translated.


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Polyadenylation of mRNA

Poly-A tail is not transcribed from DNA, but rather added after transriptionby poly-A polymerase

The tail helps to stabilize the mRNA and facilitate its exit from the nucleus.

When the poly-A mRNA enters the cytosol the tail is gradually shortened.


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tRNA modification

Addition of CCA

sequence to 3-

terminal.

This is catalyzed by

nucleotidyltransferase


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Translation: Synthesis of Proteins

Proteins are produced by the process oftranslation, which occurs on ribosomes

and is directed bymRNA.

The genetic message encoded in DNA is firsttranscribed into mRNA

The nucleotide sequence in the coding regionof the mRNA is then translated into the aminoacid sequence of the protein.


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Genetic Code


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Characteristics of Genetic code A. The Code Is Degenerate, But Unambiguous (means that an

amino acid may have more than one codon. However, each codon

specifies only one amino acid, and the genetic code is, thus,unambiguous.)

B. The Code Is Almost Universal (All organisms studied so far usethe same genetic code, with some rare exceptions. One exceptionoccurs in human mitochondrial mRNA, where UGA codes for

tryptophan instead of serving as a stop codon, AUA codes formethionine instead of isoleucine, and CUA codes for threonineinstead of leucine).

C. The Code Is Nonoverlapping and without Punctuation(mRNA does not contain punctuation to separate one codon from

the next and the codons do not overlap).


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Translation of the code Translation of the code. The portion of

mRNA that specifies the amino acidsequence of the protein is read in codons,

which are sets of three nucleotides thatspecify individual amino acids.

The codons on mRNA are read sequentiallyin the 5 to 3 direction, starting with the 5-

AUG (or start codon) that specifiesmethionine and sets the reading frameand ending with a 3-termination (or stop)codon (UAG, UGA, orUAA).

The protein is synthesized from its N-terminus to its C-terminus.

Each amino acid is carried to the ribosomeby an aminoacyl-tRNA (i.e., a tRNA withan amino acid covalently attached).

Base-pairingbetween the anticodon ofthe tRNA and the codon on the mRNAensures that each amino acid is insertedinto the growing polypeptide at theappropriate position.


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Synthesis of the protein

Initiation involves formation of a complex containing the initialmethionyl-

tRNA bound to the AUG start codon of the mRNA and to the P site of theribosome. It requires GTP and proteins known as eukaryotic initiationfactors (eIFs).

Elongation of the polypeptide involves three steps: (a) binding of anaminoacyl-tRNA to the A site on the ribosome where it base-pairs withthe second codon on the mRNA; (b) formation of apeptide bondbetween

the first and second amino acids; and (c) translocation, movement of themRNA relative to the ribosome, so that the third mRNA codon moves intothe A site.

These three elongation steps are repeated until a termination codonaligns with the site on the ribosome where the next aminoacyl-tRNA would

normally bind. Release factors bind instead, causing the completed proteinto be released from the ribosome.

After one ribosome binds and moves along the mRNA, translating thepolypeptide, another ribosome can bind and begin translation. The complexof a single mRNA with multiple ribosomes is known as apolysome.


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Several Antibiotic and Toxin inhibitors of Translation

Inhibitor Comments

Chloramphenicol inhibits prokaryotic peptidyl transferase

Streptomycin inhibits prokaryotic peptide chain initiation, also induces mRNA misreading

Tetracycline inhibits prokaryotic aminoacyl-tRNA binding to the ribosome small subunit

Neomycin similar in activity to streptomycin

Erythromycin inhibits prokaryotic translocation through the ribosome large subunit

Fusidic acid similar to erythromycin only by preventing EFG from dissociating from thelarge subunit

Puromycin resembles an aminoacyl-tRNA, interferes with peptide transfer resulting inpremature termination in both prokaryotes and eukaryotes

Diptheria toxin catalyzes ADP-ribosylation of and inactivation of eEF-2, eEF-2 contains amodified His residue known as dipthamide, it is this resudue that is the targetof diptheria toxin

Ricin found in castor beans, catalyzes cleavage of the eukaryotic large subunitrRNA

Cycloheximide inhibits eukaryotic peptidyltransferase

P t i S th i


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Protein Synthesis

DNA passes on geneticinformation so thatproteins can be

synthesized in the cell.Proteins control manydifferent chemicalprocesses in the cell such as energyproduction, intra &extracellulartransportation, enzymesynthesis, hormone

synthesis, and normalcell maintenance.

Beside is the summaryof the process from DNAreplication to RNAsynthesis, also knownas transcription, andtranslation, also known

as protein synthesis.Note that once RNA ismade in the nucleus ofthe cell, it leaves andenters the cytoplasm,where most cellularproteins are made.

dr okunowo wahab's introductory molecular biology lecture note ii

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