TRANSCRIPTION & PROTEIN SYNTHESIS

By Dr Gnanajothy Ponnudurai

LECTURE OUTLINE

A. B. B. i. ii. C. D.

TRANSCRIPTION THE GENETIC CODE PROTEIN SYNTHESIS Components required to make a polypeptide Steps involved in translation PROTEIN MATURATION AND MODIFICATION TARGETTING OF POLYPEPTIDES TO SPECIFIC DESTINATIONS IN THE CELL REGULATION OF GENE EXPRESSION IN EUKARYOTES

E.

AIMS AND OBJECTIVES:

After completing this topic you should be able to:
Describe what is transcription and post-transcriptional

modification Discuss the characteristics of genetic code Discuss the components required for protein synthesis. Describe the events that occur during protein synthesis. Explain the process of protein maturation and modification Explain the importance of regulation of gene expression in eukaryotes

OVERVIEW OF TRANSCRIPTION AND TRANSLATION

Flow of genetic information: DNA

RNA

protein
Transcription : synthesis of RNA under the

direction of DNA.
Intermediate in flow of information from DNA to

protein is messenger RNA (mRNA).

Translation : synthesis of polypeptide under the

direction of mRNA. The sites of translation are ribosomes.

Central Dogma of Molecular Biology

Replication DNA Tanscription

RNA Translation

Protein

OVERVIEW OF TRANSCRIPTION AND TRANSLATION

Eukaryotic cell: RNA is first synthesized as pre-RNA, which undergoes modifications called RNA processing before leaving the nucleus as mature RNA. Transcription occurs in nucleus whereas translation occurs in the ribosomes in cytoplasm. Prokaryotic cell : No RNA processing Transcription and simultaneously

translation

can

occur

RNA SYNTHESIS (TRANSCRIPTION OF GENES)

A closer look at Transcription
Transcription is the DNA-directed synthesis of

RNA. Transcription is catalysed by the enzyme RNA polymerase. Only one DNA strand is transcribed (copied). RNA polymerase does not require a primer.

POSTTRANSCRIPTIONAL MODIFICATION OF RNA (RNA PROCESSING)

Split genes and RNA splicing
Most eukaryotic genes are interrupted by long,

noncoding regions called introns (intervening sequences), interspread among coding regions called exons.
Both introns and exons are transcribed to form a

pre-RNA
RNA splicing involves removing the introns and

joining the exons.

THE GENETIC CODE

There are only 4 nucleotide bases to specify 20 amino

acids. 4 nucleotide bases can generate 64 possible triplet combinations. In an mRNA transcribed from a gene, these base triplets are called codons. The genetic code is a collection of codons. Of the 64 codons, 61 code for the 20 amino acids. 3 codons code for termination of protein synthesis

THE GENETIC CODE

There is redundancy in the genetic code, but no ambiguity. The genetic code is nearly universal. The start signal or initiation codon AUG sets the reading

frame. (Since AUG also stands for methionine, polypeptide chains begin with methionine when they are synthesized. An enzyme may subsequently remove this starter amino acid from a chain) The genetic code is nonoverlapping.

PROTEIN SYNTHESIS (TRANSLATION)

Translation is the RNA-directed synthesis of a polypeptide

Eukaryotic transcription and translation

DNA

Transcription

Primary transcript

pre-tRNA

pre-rRNA

pre-mRNA

Mature RNA

tRNA

rRNA

mRNA

Structural and regulatory functions. No translation

Protein

Components required to make a polypeptide

1.

Amino acids 20 different amino acids

Components required to make a polypeptide

2.

Messenger RNA (mRNA) Template for synthesis of polypeptide chain

Components required to make a polypeptide

3.

Transfer RNA (tRNA) tRNAs are transcribed from DNA templates. tRNA is the carrier of amino acid in the cytosol. A tRNA molecule has amino acid attachment site (binds and carries amino acids) and anticodon (base triplet that binds to complementary codon on mRNA)

Components required to make a polypeptide

4.

Aminoacyl-tRNA synthetases Enzymes required for the attachment of amino acids to their corresponding tRNA. The process is driven by hydrolysis of ATP.

Components required to make a polypeptide

5.

Ribosomes A ribosome consists of 2 subunits, a large subunit and a small subunit. Ribosomes = Proteins + ribosomal RNA (rRNA) Ribosome has two binding sites for tRNA molecules: A site and P site. In eukaryotes, ribosomes are either free in the cytosol or on the endoplasmic reticulum.

Components required to make a polypeptide

6.

Protein factors Initiation factors, elongation factors, release factor

Components required to make a polypeptide

7.

Energy source. For attachment of amino acid to tRNA energy is provided by ATP. For chain inititation and elongation energy is provided by GTP.

Steps involved in translation

Initiation
Formation of initiation complex

mRNA tRNA carrying Methionine (the 1st amino acid of the polypeptide) 2 subunits of ribosomes proteins called initiation factors Initiation codon (AUG) on mRNA is positioned in P site of ribosomes. Anticodon of Met-tRNA pairs correctly with initiation codon AUG.

Steps involved in translation

Elongation
tRNA carrying the 2nd amino acid binds to A site of

ribosomes. Enzyme peptidyl transferase transfers Met from its tRNA, to the second amino acid in A site, forming a peptide bond. Ribosome moves by the distance of 1 codon, towards 3-end of mRNA. tRNA without the amino acid is released back into the cytosol. Movement of ribosome shifts dipeptide-tRNA from A site to P site 3rd amino acid-tRNA binds to empty A site.

Steps involved in translation

Termination.
Termination requires stop codon. Stop codon occupies A site on ribosome. Release factor binds to A site.

Peptidyl transferase cuts completed polypeptide from tRNA

in P site. mRNA, tRNA, release factor leave the ribosome. Ribosome dissociates into subunits.

PROTEIN MATURATION AND MODIFICATION

A gene determines primary structure of a polypeptide, and

primary structure determines the three dimensional conformation.

Post-translational modification may be required before a

protein can be functional:

Disulfide bond formation and protein folding Proteolysis : Enzymes may remove one or more amino acids from the polypeptide, or cleave the polypeptide Glycosylation: certain amino acids are modified by attachment of carbodydrates Attachment of lipids Protein phosphorylation Assembly of various protein subunits

REGULATION OF GENE EXPRESSION IN EUKARYOTES

Gene expression gene is expressed when it is

transcribed and translated to yield product.

Regulation of gene expression permits only a small

fraction of total genes in a cell to be expressed at any given time. Remaining genes are inactive.

REGULATION OF GENE EXPRESSION IN EUKARYOTES

Regulation of gene expression is required for:

Adaptation cells adapt to environmental changes by turning expression of genes on and off. Development & differentiation physical and physiological changes that occur are due to variation in gene expression. Different proteins synthesised in different quantities Cells conserve energy by making proteins only when required