Biochemistry

Nucleic Acid & protein synthesis

Nucleic Acid & Protein Synthesis

Introduction & Definitions:
Nucleic acids: Large Molecules in our cells' nuclei that store and direct information for cellular growth and reproduction DNA (deoxyribonucleic acid): Genetic material in the nucleus of a cell contains al the info. Needed for the development of a complete living system RNA (ribonucleic acid): Translate the genetic info. In DNA & carries that info. To the ribosomes where the synthesis of protein takes place Genome: All genetic information in cell Genes: A segment of DNA (part) that contains the info. to make a particular protein or

Nucleic Acids (DNA & RNA) Structure:

Basic structure of DNA & RNA: Polymers of repeated monomer units called nucleotides Nucleotide structure: Nitrogenous base + 5 C (pentose) sugar (connected by glycosidic bond) nucleoside Nucleoside + phosphate group (connected to C'5 of sugar by ester bond) 5' monophosphate nucleoside = nucleotide
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Nitrogenous bases: 1) Purines (-osine): N atom in position 9 connects to C'1 of sugar

Adenine (+sugarAdenosine) Guanine (+sugarGuanosine)

2) Pyrimidine (-idine): N atom in position 1 connects to C'1 of sugar

Cytosine (+sugarCytidine) Uracil (+riboseUridine) just in RNA Thymine(+deoxyribosedeoxy-Thymidine) just in DNA

Sugar connects by its C'1 to nitrogenous base (N#1 in Pyrimidine, N#9 in

purines) by a glycosidic bond giving nucleoside

And then connects by its C'5 to phosphate group by an ester bond giving
nucleotide

#9

#1

Purine

Pyrimidine

Pentose

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Then nucleotides join together by phospho-di-ester bond between OH gp of C'3

of nucleotide #1 with phosphate gp of C'5 of nucleotide #2
Phospho-di-ester bond broken by nuclease enzyme

Nucleotide

Differences in structure of DNA & RNA:

Nitrogenous base of DNA are 2 purines adenine A & guanine G & 2 pyrimidines cytosine C & thymine T Nitrogenous base of RNA are The same except thymine substituted by uracil U The 5 C sugar in DNA is deoxyribose The 5 C sugar in RNA is ribose RNA molecules are single strand while DNA are double RNA molecules are smaller than DNA

Numbering by ' (dash) refers to sugar

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Base

Nucleoside
RNA

Nucleotide

Adenine (A) Guanine (G) Cytosine (C) Uracil (U)

Adenosine (A) Guanosine (G) Cytidine (C) Uridine (U)

Adenosine 5'-monophosphate (AMP)

Cyclic form regulates cellular fun., metabolism, & division

Guanosine 5'-monophosphate (GMP)

Cyclic form regulates cellular fun., metabolism, & division

Cytidine 5'-monophosphate (CMP) Uridine 5'-monophosphate (UMP)

*UTP used in glocuronic acid metabolism + glycogen synthesis

DNA
Adenine (A) Guanine (G) Cytosine (C) Thymine (T) Deoxyadenosine (A) Deoxyguanosine (G) Deoxycytidine (C) Deoxythymidine (T) Deoxyadenosine 5'-monophosphate (dAMP) Deoxyguanosine 5'-monophosphate (dGMP) Deoxycytidine 5'-monophosphate (dCMP) Deoxythymidine 5'-monophosphate (dTMP)

Primary structure: The sequence of bases that carries the genetic info. From one cell to the next This sequence is unique DNA double helix: 2 polynucleotide strands winding about each other like a spiral staircase The complementary base pairs of the 2 strands are connected by H bonds in a specific sequence
Adenine connects to thymine by 2 H bonds Cytosine connects to guanine by 3 H bonds (takes more time in breaking by heat) In a DNA molecule the NO. of adenine & thymine is equal Also cytosine and guanine are equal
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The 2 strands run anti-parallel to each other meaning that: One strand is in the direction 3'end5'end while The other strand is in the direction 5'end3'end DNA molecules are compact & highly coiled In chromosomes, DNA strands are wrapped around proteins called histones Which provides stability and order arrangement for the DNA double helix

Histones present inside grooves

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Factors stabilizing the structure of DNA double helix:

1) Hydrophobic bonds between N bases (N bases are hydro phobic internally, while sugar phosphate backbone is hydrophilic externally) 2) H-bonds between N bases 3) Electrostatic bonds 4) Stabilizing Prot. (Histones)

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DNA double helix has different types according to the glycosidic bond
orientation:
1) If the N base perpendicular to sugar phosphate back bone It's called B-line (most common) 2) If it's oblique DNA is called A-line 3) If it's zigzag DNA is called Z-line

A-DNA

B-DNA

Z-DNA

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Types of bonds in Nucleic acid:

Non-Covalent can be broken by heat
H-bonds Electrostatic (ionic) bonds Hydrophobic &Hydrophilic bonds

Covalent cannot be broken by heat

Glycosidic bond between pentose and N base Ester bond between pentose and phosphate group Phospho-di-ester bond between nucleotides

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DNA Replication:
Definition: Synthesizing a copy of DNA by separating the parental 2 strands and making a complementary new strand for each one of the original strands inside nucleus. Process: 1) Begins when helicase enzyme catalyzes the unwinding of a portion of the double helix by breaking the H bonds between complementary bases 2) After the base pairs are formed ,DNA polymerase catalyzes the formation of phospho-di-ester bonds between nucleotides

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Replication is a semi conservative process, in each new DNA molecule, one strand of the double helix is from the original DNA and the other is newly synthesized New DNAs are called daughter DNA and they are identical to each other and to the original one Example: Original DNA strands

TA CG AT GC

AT GC TA CG

TA CG AT GC

Semi conservative
Key: ( N- bases on the two parents(original)strands of DNA before replication) ( N-base on daughter strand and N-base on parent strand after rep) ( N-base on parent strand and N-base on daughter strand after rep) Black bases original strands Red bases newly synthesized strands The 2 old strands are called conservative

DNA

Transcription

mRNA
10

Translation

Prot.

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Transcription, RNA & Genetic code:

Types of RNA: 1) Ribosomal RNA (rRNA)
The most abundant type in cell Contained inside ribosomes which are the site of prot. Synthesis

2) Messenger RNA (mRNA)

Carries the genetic info. from the DNA in the nucleus to the ribosome for prot. synthesis Each gene (segment of DNA) produces a separate mRNA molecule in transcription The size of RNA depends on the NO. of nucleotides on the transcripted gene mRNA is broken quickly by exonucleases

3) Transfer RNA (tRNA)

Smallest of RNA molecules Translate the genetic information (codon) of mRNA to amino acid of a prot. Carries amino acid There is one or more different tRNA for each of the 20 amino acids Enzymes attach amino acid to free OH at the 3' end Consists of: I. 70-90 nucleotides II. H bonds between some of the complementary bases in the chain produce loops that give some double stranded regions III. 3' end with the nucleotide sequence ACC known as acceptor stem IV. Anticodon which is a series of 3 bases that complements 3 bases on the mRNA

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Transfer RNA
A typical tRNA molecule has an acceptor stem at the 3'end of nucleic acid where an amino acid attaches and an anticodon loop that complements three bases on mRNA

Type
rRNA mRNA tRNA

% of total RNA
75% 5-10% 10-15%

Function in the cell

Major component of the ribosome Carries info. for prot. Synthesis from DNA in the nucleus to the ribosome Brings amino acids to the ribosomes for prot. synthesis

Transcription (synthesis of mRNA): I. The section of a DNA that contains the gene to be copied unwinds II. Polymerase enzyme identifies a starting point that is the signal to begin mRNA synthesis III. C of DNA pairs with G of mRNA, A of DNA pairs with U of mRNA, T of DNA pairs with A of mRNA & G of DNA pairs with C of mRNA IV. RNA reaches the termination point that signals the end of transcription , meanwhile the new mRNA is released and the DNA returns to it's double helix structure V. This process occurs in nucleus

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Genetic code: Language that relates the codons in mRNA that specifies the a.a. order for the prot. synthesis
Language or information

Encoded in DNA nucleotides as triplet code Transcripted in mRNA nucleotides to codon Translated to amino acid by anticodon of tRNA nucleotides

Codons: Sequence of 3 bases in mRNA that specifies each amino acid in the prot.

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The codons have been detrimind for all 20 amino acids, a total of 64 codons are possible from the triplet combination of the 4 bases A, T, C&G

AUG start codon in translation & transcription UAA, UAG &UGA stop codon in translation & transcription
After transcription had finished in the nucleus, the mRNA produced will get out of the nucleus on its way to the cytoplasm to start translation.

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Translation:
Definition: The interpretation of codons in mRNA as amino acids in a peptide. This process occurs in cytoplasm. Components playing in this role:
tRNA (Transfer RNA; an RNA that places a specific amino acid into a peptide chain

at the ribosome. There is one or more tRNA for each of the 20 different amino acids.)
rRNA (a major component of ribosome) codons of mRNA Amino acids Enzymes.

All those components come together to begin translation.

Steps of translation
1. Initiation 2. Translocation 3. Termination

Initiation Stage:
When mRNA combines with smaller subunit of the ribosome in cytoplasm.

The start codon on mRNA is AUG.

The first tRNA that has the anticodon UAC will be carrying the amino acid and form

H-bonds with its complementary start codon AUG in mRNA at the (peptidal site) of the ribosome.
Constantly, all the time the type of a.a being carried by the first tRNA for the start

codon is (Methionine).
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Translocation Stage: (study with the diagram) Refer to the shift of a ribosome along mRNA from one codon (three bases) to the next codon during translation
Protein synthesis takes place on the ribosome at two near sites (Ppeptidal site) and

(aacceptor site) found on the large subunit of this ribosome.

The 2nd tRNA that holds the 2nd amino acid specific for the 2nd codon will stick to the

(acceptor site) of the ribosome.

The 1st tRNA holding the 1st amino acid which is specific for the start codon at the (p-

site) will transfer its amino acid to the 2nd amino acid of the 2nd tRNA at the (a-site). Those two amino acids will be linked by peptide bond.
Now the 1st tRNA will detach from the (p-site) and return to cytoplasm to do other

function.
Ribosome will shift to the next codon on the mRNA.

The 2nd tRNA holding both amino acids with peptide bond is now on the (p-site) after

the ribosome is shifted.

A third new tRNA approaches and will attach to the (a-site) having new amino acid

specific for the next codon.

Now the {two amino acids linked with peptide bond} on the 2nd tRNA on the (p-site)

will be transferred to the {new third amino acid with a new peptide bond} found on the third new tRNA on the (a-site), now the total amino acids found on the third tRNA is (three a.a) with two peptide bonds so a polypeptide.
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The second tRNA on the (p-site) now is empty from the two amino acids it had; after

transferring them to the third a.a.

This second tRNA will detach from the (p-site) & return to the cytoplasm.

Ribosome shift again and the process continues for the tRNAs as going through the

same sequence and same story by adding new amino acids to the polypeptide until it reaches the [stop codon] by either reading (UAG) or (UGA) or (UAA), and here the termination step occurs.

Termination:
When the ribosomes shifting reaches the stop codon after all amino acids

(polypeptide chain) had completed its sequence to form the specific protein it aims for.
There is NO tRNA to complement the termination (stop codon), so protein synthesis

ends.
An enzyme will release the completed polypeptide chain from the ribosome.

The starting a.a (Methionine) is often removed from the beginning of the peptide

chain.
The released polypeptide form new protein by taking secondary, tertiary structures.

Note: The amino acid attaches only on the acceptor stem of the tRNA (3end). Not as shown in
the following diagram, which shows that a.a attached to both stems in tRNA. As this diagram is only for simplification.
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Genetic Mutation:
Definition: A change in the DNA base sequence that alters the formation of a prot. in the cell. Factors lead to genetic mutation: 1. x-rays 2. overexposure to sun (UV sunlight radiation) 3. mutagens (chemicals) 4. Some viruses.
DNA
X-rays UV sunlight Mutagens Viruses

Alteration of DNA

Defective protein

Genetic disease (germ cells) or Cancer (somatic cells)

Types of Mutations: 1) Base substitution Mutation:

Mutation that replaces (alters) one base in DNA with a different base. Normal DNA

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Base substitution mutation

2) Frame shift Mutation:

Mutations that insert (addition) or (deletion) delete a base in DNA sequence & change all the codons after the base change.

Example:
Normal DNA ATG CAT GCA TGC ATT TCC TGC TTA AAA 1. Addition Mutation AAT GCA TGC ATG CAT TTT CCT GCT TAA Reading Frame is shifted 2. Deletion Mutation TGC ATG CAT GCA TTT CCT GCT TAA Reading Frame is shifted

Effects of Mutation: When mutation changes DNA base sequence causing change in a.a sequence, this leads to alterations in functional and structural proteins. If alteration was in functional protein, biological activity is lost.

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Genetic disease:
When protein deficiency is genetic, as its a result of defective enzyme caused by mutation in its genetic code.

E.g.: Phenylketonuria (PKU) results when DNA cannot direct the synthesis of the enzyme
phenylalanine hydroxylase, required for conversion of phenylalanine to tyrosine.

Normal: Phenylalanine
This enzyme is absent

Disease case: (PKU) Phenylalanine

But other enzymes in cells will convert into:

Tyrosine

Phenylpyruvate

Accumulation of phenylalanine & phenylpyruvate in blood lead to mental retardation.

Other some examples of genetic disease:

1. Down syndrome 2. Sickle-cell anaemia 3. Haemophilia ..etc.
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