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Macromolecular Macromolecular Synthesis:Synthesis:

Replication, Transcription Replication, Transcription & Translation& Translation

Yasmine Kanaan, PH. D.

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GeneticsGenetics

Genetics is the science of heredity

It includes the study of

what genes are how they carry information,

how they are replicated and passed to subsequent generations of cells

or passed between organisms,

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o Chromosomes: are cellular structures that physically carry hereditary information. The chromosomes contain the genes.

 o Genes: are segments of DNA (except in some viruses, in

which they are made of RNA) that code for functional products.

o Genotype: Is collection of genes Genotype of an organism is its genetic makeup. The

information that codes for all the particular characteristics of the organism.

o Phenotype: Is collection of proteinsRefers to actual, expressed properties such as the organism’s ability to perform a particular chemical reaction.

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DNADNA

As one of four macromolecules (carbohydrates, lipids, proteins, NA) in the cell, is classified as one type of NA. The other type of NA is RNA.

A very large, long molecule is made up of smaller “subunits” called Nucleotides.

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NucleotidesNucleotides

Are made up of three components:1. One of four nitrogenous bases (cyclic compounds

made up of C, H, O, N): Adenine (A), Thymine (T), Cytosine C, guanine G.

a) Purines: A and G (double-ring structure)b) Pyrimidines: T, C, and U (single-ring structure)

2. A 5-carbon sugar (deoxyribose)3. A phosphate groupThus, one base + deoxyribose +phosphate=nucleotide

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Bases are held together by Bases are held together by hydrogen bondhydrogen bond

A=T

G=C

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NucleosideNucleoside

Combination of a purine or pyrimidines plus a pentose sugar.

purine+ sugar No phosphate pyrimidine+ sugar group

N-glycosidic linkage - refers to the type of bond between the sugar and base

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DNADNAIs a double helical molecule consists of

two “strands” that form a “right-handed” spiral

The two strands are aligned antiparallel to each other (one seems to pointing downward and other upward)

The two strands are held together by hydrogen bonds.

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THE GENETIC MATERIAL

DNA1. DNA =

deoxyribonucleic acid. The sugars in DNA contain a 2' hydrogen

2. DNA is chemically stable

3. DNA functions as the carrier of genetic information (usually)

4. DNA contains the bases A, G, C, and T

RNA• RNA = ribonucleic acid.

The sugars in RNA contain a 2' hydroxyl group

• Due to the presence of the 2' hydroxyl group, RNA is less stable than DNA

• RNA usually functions as the carrier of genetic information

• RNA contains the bases A, G, C and U

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OligonucleotidesOligonucleotides

Oligonucleotide growth occurs in the 5' to 3' direction via an attack by 3' hydroxyl groups upon the 5' alpha phosphate of nucleotide triphosphates

The resulting linkage is a phosphodiester   

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Current Model for DNA ReplicationCurrent Model for DNA ReplicationThe replication of DNA is accomplished by a

multienzyme DNA replicase system.There are four stages in the replication process1. Prepriming (preparation for replication)2. Priming (synthesis of DNA primers)3. Elongation (addition of DNA sequences to the

3’-termini of primers)4. Termination (removal of RNA primers &

their replacement w/DNA sequences & the subsequent covalent joining of the DNA fragments).

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Cont1.Cont1. ReplicationReplication/ 1. Prepriming/ 1. PreprimingThe continuous unwinding of DNA at the

replication fork is performed by a number of proteins known as helicases:

1. Helicase II or III binds to the lagging strand2. The Rep protein also a helicase binds to the

leading strands.3. The subsequent stabilization of the unwound

DNA, & prevention of reanealing of the two seperated strands is provided by tetrameric aggregates of SS DNA-binding proteins (SSB).

4. During the progression of replication, the SSB proteins are recycled & bind to new single stranded sites along DNA.

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Cont2.Cont2. Replication Replication / 2. Priming/ 2. Priming

1. Involves the synthesis of the 5’-3’ RNA primers (length 10 nucleotides)

2. Priming is catalyzed by the enzyme primase

3. Six proteins dnaB, c, I, n, n’, n’’, perform specific functions & together they form an aggregate known as primosome

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Cont3.Cont3. Replication Replication / 3. Elongation/ 3. Elongation

1. Involves the addition of DNA sequences to the 3’-termini of the RNA primers synthesized during priming.

2. The chain elongation process is catalyzed by DNA polymerase III holoenzyme. The complete synthesis of an intact DNA strand is performed with two additional enzymes, DNA polymerase I & DNA Ligase.

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Cont4.Cont4. Replication Replication / 4. Termination/ 4. TerminationDuring termination degradation of RNA, &

elongation of DNA are catalyzed concurrently by two distinct active sites in DNA Polymerase I.

DNA Poly. I removes RNA primers (5’-3’ exonuclease activity).

DNA poly. I extends the DNA sequence on the 3’-terminus of one of the phosphate group at the 5’-terminus of the other.

DNA Ligase catalyzes the formation of a phosphodiester bond bet. two fragments by joining the 3’-OH terminus of one of the phosphate group at the 5’-terminus of the other.

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Cont5.Cont5. Replication Replication / 4. Termination/ 4. Termination

In animal cells and bacteriophage the energy required is provided by ATP hydrolysis. ATP

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TranscriptionTranscription

The synthesis of a single stranded RNA1. Depends on a DNA template2. Occurs in the 5’-3’ direction3. Produces an antiparallel complementary copy

of one of the two strands of the DNA4. Requires ribonucleoside triphosphates as

substrates5. Is catalyzed by RNA polymerase6. Advances by pyrophosphate cleavage, & the

subsequent hydrolysis of the pyrophosphate to inorganic phosphate.

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Cont1Cont1. Transcription. TranscriptionThe holoenzyme (2’) is required for the

synthesis of all three RNAs (mRNA, rRNA, tRNA) in E.coli.

The sigma factor () recognizes a specific binding site on the DNA strand.

The &’subunits function in the binding of the RNA poly complex to the DNA.

The binding site is called a promoterThe promoter is located about ten bp upstream

of the start of the transcription.

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Cont2Cont2. Transcription. TranscriptionThe consensus sequence (A-T-rich seq of six

bps) in the promoter serves as the recognition site for RNA poly binding. This seq is known as pribnow box in procaryotes (6bp).

In eucaryotes this seq is about 20-30 bps before the start of transcription & is called the hogness or the TATA box.

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Cont3Cont3 Termination of Transcription Termination of TranscriptionSimple termination which is due to stem-

&-loop structure in the RNA transcript that was synthesized from an inverted repeat sequence of the gene being transcribed.

Termination requiring an auxillary termination factor, a protein called rho.

rho depletes the substrate for RNA synthesis by hydrolyzing ribonucleoside triphosphates.

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TranslationTranslation

Phases in polypeptide synthesis

1. Amino acid activation

2. Initiation of polypeptide synthesis

3. Elongation of a polypeptide chain

4. Termination of polypeptide synthesis

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Cont1.Cont1. Translation Translation Polypeptide synthesis occurs on ribosomes (70 S) The 70 S ribosome of E.coli is composed of a 30 S

(small) subunit & a 50 S (large) subunit. The 30 S subunit is composed of 16 S RNA and 21

proteins. 50 S subunit contains two RNAs (23 S & 5S) and

32 proteins. Polypeptide synthesis occurs on the head & platform regions of the 30 S subunit & the upper half of the 50 S subunit.

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Cont2Cont2.. Translation TranslationThe 30 S subunit is the site of attachment

for both mRNAs and tRNAs.The peptidyl transferase site in the

central region of the 50 S subunit.

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Cont3Cont3.. TranslationTranslation1. Amino acid activation1. Amino acid activation

In the Ist step an aminoacyladenylate is synthesized by joining the carboxyl group of an amino acid w/ the alpha phosphate of ATP.

The Rxn is catalyzed by aminoacyl-tRNA synthetase & the required energy is furnished by the cleavage of pyrophosphate of ATP, & the hydrolysis of the pyrophosphate to inorganic phosphate.

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Cont4Cont4.. TranslationTranslation1. Amino acid activation1. Amino acid activation

In the 2nd step the aa of the adenylate derivative is transferred to a hydroxyl group of the 3’-terminal adenyl nuceotide of a tRNA.

The Rxn is catalyzed by aminoacyl-tRNA synthetase.

AA+ ATP+ tRNA +H20==Aminoacyl-tRNA+ AMP+ 2Pi

AA + enzyme

AAATP

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Cont5Cont5.. Translation Translation 2. Initiation of Polypeptide Synthesis2. Initiation of Polypeptide Synthesis

An initiation complex consisting of a 30 S subunit, an initiation tRNA carrying N-formylmethionine, & mRNA is formed in the initial step.

3 protein initiation factors (IF1, IF2 & IF3), & GTP are involved in this step. Formylated methionine is the aa that initiates polypeptide synthesis in E.coli.

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Cont6Cont6.. Translation Translation 2. Initiation of Polypeptide Synthesis2. Initiation of Polypeptide Synthesis

In the 2nd step of initiation, the 50 S ribosomal subunit combines w/ the 30 S initiation complex to form the 70 S initiation complex.

During this step GTP is hydrolyzed to GDP and Pi, & the three initiation factors are released from the complex.

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Cont7Cont7.. TranslationTranslationElongation of a Polypeptide ChainElongation of a Polypeptide Chain

Elongation is mRNA directed & proceeds from the N-terminus to the C-terminus of the polypeptide being synthesized.

The ist step of elongation involves the binding of an aminoacyl-tRNA/EFTu-GTP complex to the recognition (R) site of the small (30 S) subunit of a functional ribosome. EFTu is a temperature unstable elongation factor and promotes the hydrolysis of GTP to GDP and Pi.

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Cont8Cont8.. TranslationTranslationElongation of a Polypeptide ChainElongation of a Polypeptide Chain

The aminoacyl-tRNA is transferred to a second site in the 30 S subunit known as aminoacyl (A) site.

A peptide bond is formed between the two aa residues attached to the tRNAs occupying the P & A sites on the 70 S initiation complex.

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Cont9Cont9.. TranslationTranslationElongation of a Polypeptide ChainElongation of a Polypeptide Chain

Peptidyl transferase catalyzes the peptide bond formation & transfer of the N-formylmethionyl residue at the P site to the NH3 group of the aa residue attached to the aminoacyl tRNA at the A site, producing a dipeptidyl-tRNA.

The tRNA w/ out aa at the P site is released, & the peptidyl-tRNA is relocated to the P site by another elongation factor called EFG or transloase.

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Cont10Cont10.. TranslationTranslationTermination of a polypeptide bondTermination of a polypeptide bond The termination of a polypeptide synthesis is

signaled by one of the 3 termination codons (UAA, UAG, or UGA) at the end of an mRNA.

The 3-base sequence of a termination codon is recognized by one of the two release factors, RF1 (which recognizes UAA or UAG) of RF2 (which recognizes UAA or UGA).

Both release factors function in combination w/ GTP, which is hydrolyzed to GDP an Pi during termination.

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Genetic code: determines how a nucleotide sequence is converted into the sequence of protein.

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