BIOSYNTHESIS OF NUCLEIC

ACIDS AND PROTEINS

The flow of genetic information in a typical cell

DNA

RNA

protein

Primary structure of nucleic acids

Two-stranded structure of DNA

Watson JamesCrick Francis

The double helix of DNA was discovered in 1953 by Crick F. and Watson J. Nobel prize in 1962.

When replication takes place?

What are the principles of replication?

• Based on template;• Complementary;• Antiparallel;• Two directions;• Semi-conservative;• Very complex.

Components required for replications

• DNA molecule - template• Origin of replication – point ORI• Enzymes• Nucleotides (dNTP and NTP) • SSB proteins

Main enzymes required for replication• DNA-polymerase (I, II,

III – in prokaryotes,

ᵅ,ᵞ,ᵋ – in eukaryotes)

• Primase • DNA-helicases • Topoisomerase • DNA-ligase• Telomerase

Topoisomerase

Protein complexes of the replication fork

DNA replication

Reiji Okazaki provided experimental evidence for discontinuous DNA synthesis

Details of lagging strand synthesis

DNA

mRNARibosome

Polypeptide

TRANSCRIPTION

TRANSLATION

TRANSCRIPTION

TRANSLATION

Polypeptide

Ribosome

DNA

mRNA

Pre-mRNARNA PROCESSING

(a) Bacterial cell (b) Eukaryotic cell

Nuclearenvelope

TRANSCRIPTIONDNA

mRNA

(a) Bacterial cell

TRANSCRIPTIONDNA

mRNA

(a) Bacterial cell

TRANSLATIONRibosome

Polypeptide

Nuclearenvelope

DNA

Pre-mRNA

(b) Eukaryotic cell

TRANSCRIPTION

RNA PROCESSING

Nuclearenvelope

DNA

Pre-mRNA

(b) Eukaryotic cell

mRNA

TRANSCRIPTION

RNA PROCESSING

Nuclearenvelope

DNA

Pre-mRNA

(b) Eukaryotic cell

mRNA

TRANSCRIPTION

TRANSLATION Ribosome

Polypeptide

Nontemplatestrand of DNA

RNA nucleotides

RNApolymerase

Templatestrand of DNA

3

35

5

5

3

Newly madeRNA

Direction of transcription

A

A A A

AA

A

T

TT

T

TTT G

GG

C

C C

CC

G

C CC A AA

U

U

U

end

Initiation and elongationsteps of transcription

Biochemistry For Medics- Lecture Notes

26

Post Transcriptional Post Transcriptional modifications of modifications of

pre m- RNApre m- RNA• In prokaryotic organisms, the primary

transcripts of mRNA-encoding genes begin to serve as translation templates even before their transcription has been completed.

• In all eukaryotes the primary transcripts of mRNA-encoding genes undergo extensive processing before they are converted to mature functional forms

Post Transcriptional modifications of Post Transcriptional modifications of pre m- RNApre m- RNA

a) 5' Capping• Mammalian mRNA molecules contain a 7-methylguanosine cap

structure at their 5' terminal. • The cap structure is added to the 5' end of the newly transcribed

mRNA precursor in the nucleus prior to transport of the mRNA molecule to the cytoplasm.

• The 5' cap of the RNA transcript is required both for efficient translation initiation and protection of the 5' end of mRNA from attack by 5-'3' exonucleases.

• Eukaryotic m RNAs lacking the cap are not efficiently translated.

28

Post Transcriptional modifications of Post Transcriptional modifications of pre m- RNApre m- RNA

•The addition of the Guanosine triphosphate (part of the cap is catalyzed by the nuclear enzyme guanylyl transferase.

•Methylation of the terminal guanine occurs in the cytoplasm. and is catalyzed by guanine-7-

methyl transferase.•S-Adenosyl methionine is the methyl group donor. •Additional methylation steps may occur.The secondary methylations of mRNA molecules, those on the 2'-hydroxy and the•N6 of adenylyl residues, occur after the mRNA molecule has appeared in the cytoplasm

29

Post Transcriptional modifications of Post Transcriptional modifications of pre m- RNApre m- RNA

b) Addition of poly A tail• Poly(A) tails are added to the 3' end of mRNA molecules in a

posttranscriptional processing step. • The mRNA is first cleaved about 20 nucleotides downstream from an

AAUAA recognition sequence • Another enzyme, poly(A) polymerase, adds a poly(A) tail which is

subsequently extended to as many as 200 A residues. • The poly(A) tail appears to protect the 3' end of mRNA from 3' 5'

exonuclease attack. • Histone and interferon's mRNAs  lack poly A tail.•  After the m-RNA enters the cytosol, the poly A tail is gradually shortened.

30

Post Transcriptional modifications of Post Transcriptional modifications of Pre m RNAPre m RNA

Removal of introns  (Splicing)• Introns or intervening sequences are the

RNA sequences which do not code for the proteins.

• These introns are removed from the primary transcript in the nucleus, exons (coding sequences) are ligated to form the mRNA molecule, and the mRNA molecule is transported to the cytoplasm.

• The steps of splicing are as follows-32

Post Transcriptional modifications of Post Transcriptional modifications of Pre m RNAPre m RNA

• Introns are removed from the primary transcript in the nucleus, exons (coding sequences) are ligated to form the mRNA molecule

33

Figure 17.12-1RNA transcript (pre-mRNA)

5Exon 1

Protein

snRNA

snRNPs

Intron Exon 2

Other proteins

Figure 17.12-2RNA transcript (pre-mRNA)

5Exon 1

Protein

snRNA

snRNPs

Intron Exon 2

Other proteins

Spliceosome

5

Figure 17.12-3RNA transcript (pre-mRNA)

5Exon 1

Protein

snRNA

snRNPs

Intron Exon 2

Other proteins

Spliceosome

5

Spliceosomecomponents

Cut-outintronmRNA

5Exon 1 Exon 2

Nirenberg Marshall decoded the genetic code. Nobel prize, 1968

GENETIC CODE - sequence of mononucleotides in mRNA that specifies the sequence of amino acids in peptide chain

CODON – mRNA triplet base sequence responsible for 1 amino acid

PROPERTIES OF GENETIC CODE

1. Degenerate

2. Specific

3. Nonoverlapping

4. Without punctuation

5. Universal

TRANSLATION

• 1. Recognition

• 2. Initiation

• 3. Elongation

• 4. Termination

Formation of aminoacyl tRNAs by aminoacyl tRNA synthetase.

RECOGNITION

Aminoacyl-tRNAsynthetase (enzyme)

Amino acid

P P P Adenosine

ATP

Figure 17.16-1

Aminoacyl-tRNAsynthetase (enzyme)

Amino acid

P P P Adenosine

ATP

P

P

P

PPi

i

i

Adenosine

Figure 17.16-2

Aminoacyl-tRNAsynthetase (enzyme)

Amino acid

P P P Adenosine

ATP

P

P

P

PPi

i

i

Adenosine

tRNA

AdenosineP

tRNA

AMP

Computer model

Aminoacid

Aminoacyl-tRNAsynthetase

Figure 17.16-3

Aminoacyl-tRNAsynthetase (enzyme)

Amino acid

P P P Adenosine

ATP

P

P

P

PPi

i

i

Adenosine

tRNA

AdenosineP

tRNA

AMP

Computer model

Aminoacid

Aminoacyl-tRNAsynthetase

Aminoacyl tRNA(“charged tRNA”)

Figure 17.16-4

Components of a 70S prokaryotic ribosome                                                          

tRNAmolecules

Growingpolypeptide Exit tunnel

E PA

Largesubunit

Smallsubunit

mRNA5

3

(a) Computer model of functioning ribosome

Exit tunnel Amino end

A site (Aminoacyl-tRNA binding site)

Smallsubunit

Largesubunit

E P AmRNA

E

P site (Peptidyl-tRNAbinding site)

mRNAbinding site

(b) Schematic model showing binding sites

E site (Exit site)

(c) Schematic model with mRNA and tRNA

5 Codons

3

tRNA

Growing polypeptide

Next aminoacid to beadded topolypeptidechain

Figure 17.17

Figure 17.17a

tRNAmolecules

Growingpolypeptide Exit tunnel

E P A

Largesubunit

Smallsubunit

mRNA5

3

(a) Computer model of functioning ribosome

Figure 17.17b

Exit tunnel

A site (Aminoacyl-tRNA binding site)

Smallsubunit

Largesubunit

P A

P site (Peptidyl-tRNAbinding site)

mRNAbinding site

(b) Schematic model showing binding sites

E site (Exit site)

E

Figure 17.17c

Amino end

mRNAE

(c) Schematic model with mRNA and tRNA

5 Codons

3

tRNA

Growing polypeptide

Next aminoacid to beadded topolypeptidechain

Initiation of protein biosynthesis in prokaryotes.

Elongation of the Polypeptide Chain

• During the elongation stage, amino acids are added one by one to the preceding amino acid at the C-terminus of the growing chain

• Each addition involves proteins called elongation factors and occurs in three steps: codon recognition, peptide bond formation, and translocation

• Translation proceeds along the mRNA in a 5′ to 3′ direction

Amino end ofpolypeptide

mRNA

5

E

Psite

Asite

3

Amino end ofpolypeptide

mRNA

5

E

Psite

Asite

3

E

GTP

GDP P i

P A

Amino end ofpolypeptide

mRNA

5

E

Psite

Asite

3

E

GTP

GDP P i

P A

E

P A

Elongation1) Positioning of aminoacyl-tRNA in the A site 2) Formation of the peptide bound (enzyme – peptidyl transferase)3) Translocation

Amino end ofpolypeptide

mRNA

5

E

Asite

3

E

GTP

GDP P i

P A

E

P A

GTP

GDP P i

P A

E

Ribosome ready fornext aminoacyl tRNA

Psite

Termination of translation in prokaryotes

POSTTRANSLATIONAL MODIFICATION

1) Preparing of proteins for different functions

2) Direction of proteins to different locations (targeting)

1. Proteolytic cleavage

2. Hydroxylation

3. Glycosilation

4. Phosphorilation

5. Lipophilic modification

The operon model (by Jacob and Monod)

Some inhibitors of transcription

Some antibiotics that act by interfering with protein biosynthesis