Molecular Biology IIBCH 446

Dr. Amina R ELGezeery Biochemistry Dept

King Saud University

Continuous Assessment Tests (CAT)

• • Two Tests --------------------------40 Marks • • Two quiz --------------------------10 Marks • • Final----------------------------------50 Marks

Dates for CAT : – 1st CAT: … Sunday 23 Dhu-Al-Qadah 1431 – 2nd CAT: … Tuesday 8 Muharram 1432

• Time: 1.00-2.00• Lecture Room: B 8 / R 686

Ref. Books

• • Lehninger: Pronciples of Biochemistry by DL. Nelson and MI. Cox .

• From genes to genomes . Dale J W and von Schantz M .

Outline of lectures 1&2

• Introduction to molecular biology :• DNA as a genetic material.• DNA Structure and characters

Molecular Biology

• Molecular biology is the study of gene structure and functions at the molecular level to understand the molecular basis of hereditary , genetic variation, and the expression patterns of genes .

• The molecular biology field overlaps with other areas , particularly genetic , biochemistry ,bacteriology and cell biology .

What is the Molecule of Hereditary ?

Proteins? DNA? RNA?

•Griffith’s Experiment •Avery and Macleod Experiment •Hersey and ChaseExperiment

• DNA was discovered in 1869 by Friedrich Miescher as a new, acidic, phosphorus containing substance made up of very large molecules that he named “nuclein”, but its biological role was not recognized.

In 1889 Richard Altmann introduced the term “nucleic acid.”

By 1900 the purine and pyrimidine bases were known. Twenty years later, the two kinds of nucleic acids, RNA and DNA, were

distinguished.

• In (1928)and (1944) it was indicated that DNA .could be the carrier of genetic information .

Griffith’s Experiment

Griffith’s Experiment

Avery, MacLeod, and McCarty (1944) , elucidated the chemicalbasis of the transforming principle. From cultures of an S strain (1) they produced an extract of lysed cells (cell-free extract) (2). After all its proteins, lipids, and polysaccharides had been removed, the extract still retained the ability to transform pneumococci of the R

strain to pneumococci of the S strain (transforming principl) (3) .

Avery and co-workers determined that this was attributed to the DNA alone. Thus, the DNA must contain the correspondinggenetic information.

This explained Griffith’s observation. Heat inactivation had left the DNA of the bacterial chromosomes intact. The section of the chromosome with the gene responsible for capsule formation (S gene) could be released from the destroyed S cells and be taken up by some R cells in subsequent cultures. After the S gene was incorporated into its DNA, an R cell was transformed into an S cell (4).

The Transforming PrincipleExperiment of Avery, Macleod and McCarty (1944)

The final evidence that DNA, and no other molecule, transmits genetic information was provided by Hershey and Chase in 1952. They labeled the capsular protein of bacteriophages with radioactive sulfur (35S) and the DNA with radioactive phosphorus (32P). When bacteria were infected with the labeled bacteriophage,

only 32P (DNA) entered the cells, and not the 35S (capsular protein). The subsequent formation of new, complete phage particles inthe cell proved that DNA was the exclusive carrier of the genetic information needed to form new phage particles,

including their capsular protein .

The Hershey and Chase Experiment

‘DNA is the carrier of genetic informations’

Diagram of T2 bacteriophage injecting its DNA into an E. coli cell.

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84

DNA as the Carrier of Genetic Material

• Any substance which form the heriatable material must fulfill some essential requirements and DNA was found to fulfill them .

• 1- It is stable .• 2- It is able to carry and transcribe information which are

required to control the processes which give the organism its specificity .( transcription )

• 3- It is capable of replicating exactly, so that the genetic determinants are transmitted down from cell to cell and from generation to generation unchanged .

• 4- It is able to mutate .

DNA

•–Location –Structure –Biosynthesis –Function –Synthesis of RNA –Gene Expression

Genome: entire complement of DNA molecules of each organism

Overall function of genome:

-Control the generation of molecules (mostly RNA & proteins) that will regulate the cell function and structure . - Transfer the genetic information from cell to cell ( cell division ) and from generation to generation

without change.

DNA :

Double helix

Stores genetic code as a linear sequence of bases

≈ 20 Å in diameter

Human genome ≈ 3.3 x 109 bp

≈ 25,000 genes

General structural features

The DNA Double Helix

The double-bonded structure is stabilized by

1. Hydrogen bonding between complementary bases A bonded to T by two hydrogen bonds C bonded to G by three hydrogen bonds

2. Base stacking Within the DNA, the bases are oriented so that the flattened

regions are facing each other

General structural features

The DNA Double Helix

There are two asymmetrical grooves on the outside of the helix

1. Major groove

2. Minor groove

Certain proteins can bind within these grooves They can thus interact with a particular sequence of bases

General structural features

The DNA Double Helix

Two strands are twisted together around a common axis

There are 10 bases per complete twist The two strands are antiparallel

One runs in the 5’ to 3’ direction and the other 3’ to 5’ The helix is right-handed

As it spirals away from you, the helix turns in a clockwise direction

A pairs with T (2 H-bonds)

G pairs with C (3 H-bonds)

Schematic model

Space-filling model

Coding strand 5’→ 3’ .Non-coding strand 3’ → 5’ .

“Chargaff’s rules”

1 .The base composition of DNA generally varies from one species to another.

2 .DNA specimens isolated from different tissues of the same species have the same base composition.

3 .The base composition of DNA in a given species does not change with an organism’s age,nutritional state, or changing environment.

4 .In all cellular DNAs, regardless of the species, the number of adenosine residues is equal to the number of thymidine residues (that is, A T) ,and the number of guanosine residues is equal tothe number of cytidine residues (G C). From these relationships it follows that the sum of the purine residues equals the sum of the pyrimidineresidues; that is, A + G= T + C.

Nucleic acid structure can be described in terms of hierarchical levels of complexity as primary, secondary and tertiary structures. -The primary structure of a nucleic acid is its covalent structure and nucleotide sequence.

- Any regular, stable structure taken up by some or all ofthe nucleotides in a nucleic acid can be referred to assecondary structure ( eg .Double helex ) . -The complex folding of large chromosomeswithin eukaryotic chromatin and bacterial nucleoids is .generally considered tertiary structure

DNA wound around histone

proteins

•Gives Maximum Absorption at OD 260. •Denaturation: dsDNA ssDNA •Melting Temperature (Tm): Temperature at which 50% of the dsDNA is changed to ssDNA. Depends on GC content. •Hyperchromiceffect: on denaturationthe OD260 increases. •Hypochromic effect: on renaturation the OD260decreases. •OD260/OD280 ratiois around 2 for pure DNA sample. •Extinction Co-efficientof DNA: 20 for 1mg/ml DNA( The Beer-Lambert Law can be used for calculation of DNA concentration: A= ΕxCxl ).

• Why the absorbance of ssDNA is higher than that of dsDNA ?

• Is DNA denaturated in vivo ? Explain

The DNA double helix can form different types of secondary structure

The predominant form found in living cells is B-DNA

However, under certain in vitro conditions, A-DNA and Z-DNA double helices can form

Different Structural Forms Of Nuclear DNA

*The B form is the most stable structure for a random-sequence DNA molecule under physiological conditions and is therefore the standard point of reference in any study of the properties of DNA.

B-DNA

Right-handed helix 10 bp per turn Most stable form .

A-DNA Right-handed helix 11 bp per turn Occurs under conditions of low humidity Little evidence to suggest that it is biologically important

-The reagents used to promote crystallization of DNA tend to dehydrate it, and thus most short DNA molecules tend to crystallize in the A form. Whether A-DNA occurs in cells is uncertain .

Z-DNA Left-handed helix

12 bp per turn The DNA backbone takes on a zigzag appearance . The major groove is barely apparent in Z-DNA, and the minor groove is narrow and deep. There is evidence for some short stretches (tracts) of Z-

DNA

Its formation is favored by GG-rich sequences, at high salt concentrations Cytosine methylation, at low salt concentrations

Evidence from yeast suggests that it may play a role in transcription and recombination

Right Handed B & A forms Left Handed Z form

Bases substantially tilted relative to the central

axis

Bases substantially tilted relative to the central

axis

Sugar-phosphate backbone follows a

zigzag pattern

Bases relatively perpendicular to the

central axis

Certain DNA Sequences Adopt Unusual Structures

A number of sequence-dependent structural variations have been detected within larger chromosome that may affect the function and metabolism of the DNA segments

1 -Bends occur in the DNA helix wherever four or more adenosine residues appear sequentially in one strand.Six adenosines in a row produce a bend of about 18.

The bending observed with this and other sequences maybe important in the binding of some proteins to DNA.

2 -palindrome Sequence :A palindrome is a word, phrase, or sentence that is spelled identically read either forward or backward ;eg.

ROTATOR. The term is applied to regions of DNA with inverted repeats of base sequence having twofold symmetryover two strands of DNA . Such sequences are self-complementary within each strand and therefore have the potential to form hairpin or cruciform(cross-

shaped) structures .

Inverted repeat can lead to loop formation

Also called hair-pin

Complementary regions

Noncomplementary regions

Held together by hydrogen bonds

Have bases projecting away from double stranded regions

DNA cruciform

Holliday junction

Sequences of these types are found in virtually every large DNA molecule and can encompass a few base pairs or thousands. The extent to which palindromes occur as cruciforms in cells is not known, although some cruciform structures have been demonstrated in vivo in E.coli. Self-complementary sequences cause isolated single strands of DNA (or RNA) in solution to fold into complex structures containing multiple hairpins.

3 -Mirror repeat sequence : When the inverted repeat occurs within each individual strand of the DNA, the sequence is called a mirror repeat.

Mirror repeats do not have complementary sequenceswithin the same strand and cannot form hairpin or cruciform structures.

4 -H-DNA : It is found in polypyrimidine or polypurine tract that also incorporate a mirror repeat.

A simple example is a long stretch of alternating T and C residues. The H-DNA structure features the triple-stranded. Two of the three strands in

the H-DNA triple helix contain pyrimidines and the third contains purines.

(a ) A sequence of alternating T and C

residues

(b )These sequences form an unusual structure in which the strands in one half of the mirror repeat are separated and the pyrimidine containing strand (alternating T and C residues) folds back on the otherhalf of the repeat to form a triple helix. The purine strand (alternating A and G residues) is left unpaired. This structure produces a sharpbend in the DNA.

H DNA

In the DNA of living cells, sites recognized by manysequence-specific DNA-binding proteins are arranged as palindromes, and polypyrimidine or polypurine sequences that can form triple helices or even H-DNA ,are found within regions involved in the regulation

of expression of some eukaryotic genes .

Synthetic DNA strands designed to pair with these sequences to form triplex DNA could disrupt geneexpression. This approach to controlling cellular metabolism is of growing commercial interest for its potential application in medicine and agriculture.

In the late 1950s, Alexander Rich et al discovered triplex DNA It was formed in vitro using DNA pieces that were made

synthetically

In the 1980s, it was discovered that natural double- stranded DNA can join with a synthetic strand of DNA to form triplex DNA The synthetic strand binds to the major groove of the

naturally-occurring double-stranded DNA

DNA Can Form a Triple Helix

- A cytidine residue (if protonated) can pair with the guanosine residue of a GC nucleotide pair, and a thymidine can pair with the adenosine of an AT pair . - The N-7, O6, and N6 of purines, the atoms that participate in the hydrogen bonding of triplex DNA,are often referred to as Hoogsteen positions, and the non-Watson-Crick pairing is called Hoogsteen pairing . - The triplexes are most stable at low pH . - The triplexes form most readily within long sequences containing only pyrimidines or only purines in a given strand. - Some triplex DNAs contain two pyrimidine strands and one purine strand; others contain two purine strands andone pyrimidine strand.

Triplex DNA formation is sequence specific

The pairing rules are

Triplex DNA has been implicated in several cellular processes

Replication, transcription, recombination

Cellular proteins that specifically recognize triplex DNA have been recently discovered

T binds to an AT pair in

biological DNA

C binds to a CG pair in

biological DNA

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62Bond representation of triplex DNA. This view is down the long axis. The “third” strand is colored.

Four DNA strands can also pair to form a tetraplex(quadruplex), but this occurs readily only for DNA sequences with a very high proportion of guanosineresidues The guanosine tetraplex, or G tetraplex, is quite stable over a wide range of conditions.

triplex and even quadruplex pairing can take place. These structures are critical for the proper replication of chromosomal DNA and repair of damaged DNA.

Also there is a tendency for many of these unusual structures to appear at sites where important events in DNA metabolism (replication, recombination, transcription) are initiated or regulated

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Quadruplex DNA