Introduction to molecular biologybiology

Dr Saeb Aliwaini

Course description

• The principal aim of the course is to equipstudents with a basic knowledge of themolecular biology of the cells

Course description

• Basic properties of cells, techniques used in celland molecular biology, the structure andfunction of the nucleus, Genes andchromosomes, DNA replication, transcription,translation, cell signalling.translation, cell signalling.

• This will be linked to diseases and its diagnosisand treatment.

Course description

• For example , what is cancer how do wediagnose and treat.

Week topic 1 The Molecular Nature of Genes :2 Gene Function3 From gene to protein4 DNA replication and telomere maintenance5 Cell cycle control , apoptosis, autophagy6 DNA repair and recombination6 DNA repair and recombination7 Cancer8 Molecular diagnostics 9 Molecular Therapeutics

Grades: Midterm Exams 30%Final Exam 60%Quizzes 10% Total = 100 %Total = 100 %

The Nature of GeneticMaterial

• DNA and RNA

• RNA is composed of a sugar (ribose) plus four nitrogen-containing bases, and that DNA contains a different sugar(deoxyribose) plus four bases.

• Each base is coupled with a sugar–phosphate to form a• Each base is coupled with a sugar–phosphate to form anucleotide

• Streptococcus pneumoniae

• A spherical cell surrounded by a mucous coat called a capsule

Somehow the virulent trait passed from the deadcells to the live, avirulent ones.

The missing piece of the puzzle was the chemical nature ofthe transforming substance

Genes are made from DNA

• Later it was found that the enzyme deoxyribonuclease (DNase),

which breaks down DNA, destroyed the transforming ability of

the virulent cell extract. These results suggested that the

transforming substance was DNA.transforming substance was DNA.

• Confirmed by:

• Ultracentrifugation

• Electrophoresis

• Ultraviolet Absorption Spectrophotometry

• Yet, by 1953, when James Watson and Francis Crickpublished the double-helical model of DNA structure,most geneticists agreed that genes were made ofDNA.

Genes are made from DNA

• Bacteriophage (bacterial virus) called T2 that infectsthe bacterium Escherichia coli.

• The phage genes enter the host cell anddirect the synthesis of new phage particles

• The phage is composed of protein and DNA only.

• The question is this: Do the genes reside in theprotein or in the DNA?

on infection, most of the DNA

Genes are made from DNA

on infection, most of the DNAentered the bacterium, along withonly a little protein. The bulk of theprotein stayed on the outside

Other experiments showed that some viral genesconsist of RNA.

The Chemical Nature of Polynucleotides

• bases, phosphoric acid, and the sugar deoxyribose (hence thename deoxyribonucleic acid).

• The four bases found in DNA are adenine (A), cytosine (C),guanine (G), and thymine (T).

• Adenine and guanine are related to the parent molecule,purine.

Primes are used in thenumbering of the ringpositions in the sugars todifferentiate them fromthe ring positions of thebases. Hydrogen andcarbon atoms are usuallyomitted for clarity.

• Nitrogen-containing molecules having the chemical properties ofa base (a substance that accepts an H+ ion or proton in solution).

Nitrogenous bases

• Chargaff ’s rules:- Using chromatography that- Using chromatography that

- [A]=[T] conc

- [G]=[C] conc

- And purine bases equals that of the pyrimidine bases ([A]+[G]=[T]+[C])

- “percent G+C,” differs among species but is constant in all cells of anorganism within a species. The G+C content can vary from 22 to 73%,depending on the organism.

- RNA contains a pentose (five-carbon) sugar called ribose.

- DNA contain the sugar deoxyribose.

- Both sugars have an oxygen as a member of the five-member ring.

- The 5′-carbon is outside the ring.

- Sugars differ only in the presence or absence (“deoxy”) of anoxygen in the 2′ position.(2′-hydroxyl group)

Sugars

The base is linked to the 1′-positionof the sugar= nucleoside

Nucleotides are nucleosides with aphosphate group attached througha phosphoester bond

• (PO4) gives DNA and RNA the property of an acid

• (a substance that releases an H+ ion or proton in solution)“nucleic acid.”

• esters linking give stabilization

• after the phosphodiester bond is formed, one oxygen atom ofthe phosphate group is still negatively ionized

The phosphate functional group

the phosphate group is still negatively ionized

• The negatively charged phosphates are extremely insoluble inlipids. How does this help?

An ester is an organic compound formed from an alcohol (bearinga hydroxyl group) and an acid

These are calledphosphodiester bondsbecause they involvephosphoric acid linked to twosugars: one through a sugar5’-group, the other through asugar 3’-group

The top of the molecule bearsa free 5’-phosphate group, so itis called the 5’-end. Thebottom, with a free 39-hydroxyl group, is called the 3’-end

Significance of 5′ and 3′

• This 5′→3′ directionality of a nucleic acid strand is an extremelyimportant property of the molecule.

• By convention, a DNA sequence is written with the 5′ end to theleft, and the 3′ end to the right.

• The number of base pairs (bp) is used as a measure of length of a• The number of base pairs (bp) is used as a measure of length of adoublestranded DNA. kilobase pair (kb or kbp) OR (Mb or Mbp)

• (usually less than 50 bases) called oligonucleotides.

• This lecture will focus on DNA. Various chemical forces drive theformation of the DNA double helix. These include hydrogen bondsbetween the bases and base stacking by hydrophobic interactions.

Hydrogen bonds

• Between the nitrogenous bases on opposite strands of theinterwound DNA chains.

• Hydrogen bonds are very weak bonds that involve the sharing ofa hydrogen between two electronegative atoms, such as oxygenand nitrogen.

• The hydrogen bonding between bases is referred to as “Watson–Crick” or “complementary” base pairing.

• adenine (A) normally pairs with thymine (T) by two hydrogenbonds, and guanine (G) pairs with cytosine (C) by three hydrogenbonds.

• There are proofreading mechanisms and DNA repairmechanisms that recognize non Watson–Crick base pairs andcorrect the majority of mistakes

• G-U base pairing is stable, and is of importance in RNA structureand RNA–protein interactions

Base stacking provides chemical stability to the DNA double helix

• The nitrogenous bases are hydrophobic nonpolar.

•

• Once the bases are attached to a sugar and a phosphate toform a nucleotide, they become soluble in water, but even sotheir insolubility still places strong constraints on the overalltheir insolubility still places strong constraints on the overallconformation of DNA in solution.

• The paired, relatively flat bases tend to stack on top of oneanother by means of a helical twist

A double-stranded DNA molecule thus has a hydrophobic corecomposed of stacked bases, the sugars and phosphates are soluble in water they orient towards the outside of the helix .

Structure of the Watson–Crick DNA double helix

• polarity in each strand (5′→3′) of the DNA double helix: one endof a DNA strand will have a 5′-phosphate and the other end willhave a 3′-hydroxyl group

• Watson and Crick found that hydrogen bonding could only occur• Watson and Crick found that hydrogen bonding could only occurif the polarity of the two strands ran in opposite directions

• The DNA double helix is also referred to as double-stranded DNA(dsDNA) or duplex DNA to distinguish it from the single-strandedDNA (ssDNA) found in some viruses

DNA has a structure verysimilar to the one justdescribed, but the helixcontains about 10.4 bp perturn.

• The sugar–phosphate backbone is not equally spaced and resultsin what are called the major and minor grooves of DNA.

• Two grooves of different width—a wider major groove and a morenarrow minor groove—that spiral around the outer surface of thedouble helix.

• The major groove carries a message (the base sequence of the• The major groove carries a message (the base sequence of theDNA) in a form that can be read by DNA-binding proteins.

• most transcription factors (proteins involved in regulating geneexpression) bind DNA in the major groove.

• B-DNA is a right-handed helix; it turns in a clockwise mannerwhen viewed down its axis

• B-DNA occurs under conditions of high humidity (95%) andrelatively low salt

• The predominant form in vivo is B-DNA.

• If the water content is decreased and the salt concentrationincreased (A-DNA) will occur

• If the water content is decreased and the salt concentrationincreased (A-DNA) will occur

• the bases are tilted with respect to the axis and thereare more(11) bases per turn than in B-DNA

• backbone formed a zig-zag structure, they called the structureZ-DNA

• A left-handed helix turns counterclockwise

DNA can undergo reversible strand separation

• During DNA replication and transcription, the strands of the helixmust separate transiently and reversibly.

• This equals denaturation in Vitro = unwinding

• Heating lead to broken hydrogen bonds, whereas thephosphodiester bonds remain intact .phosphodiester bonds remain intact .

• As DNA denatures, its absorption of UV light increases, aphenomenon known as “hyperchromicity.”

• The temperature at which half the bases in a double-strandedDNA sample have denatured is denoted the melting temperature(Tm)

Hyperchromic shift

• The amount of strand separation, or melting, is measured bythe absorbance of the DNA solution at 260 nm.

• Nucleic acids absorb light at this wavelength because of theelectronic structure in their bases.

• But when two strands of DNA come together, the close• But when two strands of DNA come together, the closeproximity of the bases in the two strands quenchessome of this absorbance.

• When the two strands separate, this quenching disappearsand the absorbance rises 30–40%

• Heating is not the only way to denature DNA. Organicsolvents such as dimethyl sulfoxide and formamide, or highpH, disrupt the hydrogen bonding between DNA strandsand promote denaturation.

• When heated solutions of denatured DNA are slowly cooled, singlestrands often meet their complementary strands and form a newdouble helix. This is called “renaturation” or “annealing.”

• Lowering the salt concentration of the DNA solution also aidsdenaturation by removing the ions that shield the negative chargeson the two strands from each other.

• At very low ionic strength, the mutually repulsive forces of thesenegative charges are strong enough to denature the DNA at arelatively low temperature.

• The G+C content of a DNA molecule has a significant effect onits Tm because GC base pair has three hydrogen bonds

The melting temperature alsodepends on the salt concentration: inlow salt, a given DNA will melt at alower temperature than in a highersalt concentration.This is because DNA is a polyanionicmolecule. The salt "shields" themolecule. The salt "shields" thenegative charges on each phosphate.When the charges are NOT shielded,the electrostatic repulsion makes itenergetically more favorable toseparate the strands.

• Also high pH or organic solvents such as formamide disrupt• Also high pH or organic solvents such as formamide disruptthe hydrogen bonding between DNA strands and promotedenaturation.

Unusual DNA secondary structures

• Slipped structures :

5′-TACGTACGTACGTACG-3′

A tandem repeat (sometimes called a direct repeat) usually upstreamof regulatory sequences

Cause DNA to assume unusual DNA secondary structures and blockingreplication forks and promoting repair

Friedreich’s ataxia

The disorder is caused by a 5′-GAA-3′ trinucleotide repeat

expansion in the first intron of the Friedreich’s ataxia gene,

which is located on chromosome 9

the earlier the onset of thedisease and the quicker the decline of the patient.

Friedreich’s ataxia

• Normally, the gene may be expanded up to ~33 repeats (GAAtrinucleotide repeats), but when the sequence is longer than59 repeats it appears to alter the architecture of the DNAsequence by causing the usual double-helix structure to foldsequence by causing the usual double-helix structure to foldback on itself into a triplex formation (‘sticky DNA') (Sakamotoet al 1999). This interferes with transcription of the gene,resulting in a deficiency of the protein encoded in that gene,in this case named frataxin (Bidichandani SI, et al 1998).Increasing length of the repeat expansion correlates withgreater reduction in levels of frataxin and increased severityof the disorder (Campuzano et al 1997).

• Cruciform structures

Experimental evidence has led to the hypothesis that cruciform structurescan act as regulatory elements in DNA replication and gene expression invarious prokaryotic and eukaryotic systems. Confirmation of a functionalrole in vivo awaits further investigation.

The DNA becomes rearranged so each repeat pairs with the complementary sequence on its own strand of DNA, instead of with thecomplementary sequence on its own strand of DNA, instead of with thecomplement on the other strand.

Triple helix DNA

Tertiary structure of DNA

• Many naturally occurring DNA molecules are circular, with nofree 5′ or 3′ end.

• The 5′ end of one strand can only join its own 3′ end tocovalently close a circle.covalently close a circle.

• Two circles of single-stranded DNA twisted around each other

• This DNA can then become supercoiled, supercoils are atwisted, three-dimensional structure which is more favorableenergetically

linking number (L) of 10

the result is an energetically relaxed circle that lies flat

underwound by one full turn to the left and then theends are sealed together,

If L=9

the result is a strained circle

ends are sealed together,

• Generally, changes in the average number of base pairs perturn of the double helix will be counteracted by the formationof an appropriate number of supercoils in the oppositedirection

• Overtwisting of the double helix usually leads to positive(right-handed)

• one negative (left-handed)

• For example, if the double helix is overwound by one full turnto the right and then the ends are sealed together the result isa strained circle with 9.5 bp/turn.

• The supercoiled state is inherently less stable than relaxedDNAThe supercoiled state is inherently less stable than relaxedDNA

• The stress present within supercoiled DNA moleculessometimes leads to localized denaturation

• After replication and transcription

Topoisomerases relax supercoiled DNA

• Topological isomers (topoisomers)

• Topoisomers can be visualized by their differing mobilitieswhen separated by gel electrophoresis

• Both type I and type II topoisomerases play important roles inmany cellular processes, including chromosome condensationand segregation, DNA replication, gene transcription, andrecombination

• Topoisomerases are highly conserved enzymes that convert(isomerize) one topoisomer of DNA to another

• type I and type II

• Type I topoisomerases are proficient at relaxing supercoiledDNA

• They act by forming a transient single-stranded break in theDNA (cleavage of a phosphodiester bond between adjacentnucleotides) and, while winding the broken ends, pass theother strand through the break

Topoisomerase-targeted anticancer drugs