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TRANSCRIPTIONAND TRANSLATION

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SIGNIFICANCE OF TRANSCRIPTION AND TRANSLATION IN MEDICINE

• Example:

• Inhibitor of RNA polymerase II: α-amanitin• Poisoning: toadstool (Amanita phalloides)

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TRANSCRIPTION AND TRANSLATION:

Transcription:

1. Essence of transcription

2. RNA polymerase

3. Mechanism of transcription

4. Posttranscriptional processing

5. RNA splicing

Translation:

6. Essence of translation

7. Structure and function of ribosome

8. Structure and function of tRNA

9. Mechanism of translation

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1. ESSENCE OF TRANSCRIPTION:

Transcription: synthesis of new complementary RNA strand according

to DNA template (copying genetic information from DNA).

Synthesis of new RNA strand is catalyzed by RNA polymerase. [FIG.]

During transcription, one strand of DNA double helix serves as a

template (template strand) while the second strand is referred to as

coding strand.

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2. RNA POLYMERASE:

RNA polymerase: it catalyzes the formation of phosphodiester bond

between subsequent ribonucleotides.

Properties of RNA polymerase:

It has only 5´→3´ polymerase activity (similarly like DNA polymerase)

according to template in 3´→5´ direction.

It is able to start the synthesis of new RNA strand (on the contrary to DNA

polymerase). [FIG.]

3 types of RNA polymerase in eucaryotic cell:• RNA polymerase I: genes for most of rRNAs (with the exception of 5S

rRNA)• RNA polymerase II: all genes encoding proteins, genes of some small

RNAs• RNA polymerase III: genes encoding tRNAs, gene for 5S rRNA, genes

of some small RNAs

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2. RNA POLYMERASE:

RNA polymerase: it catalyzes the formation of phosphodiester bond

between subsequent ribonucleotides.

Properties of RNA polymerase:

It has only 5´→3´ polymerase activity (similarly like DNA polymerase)

according to template in 3´→5´ direction.

It is able to start the synthesis of new RNA strand (on the contrary to DNA

polymerase). [FIG.]

3 types of RNA polymerase in eucaryotic cell:• RNA polymerase I: genes for most of rRNAs (with the exception of 5S

rRNA)• RNA polymerase II: all genes encoding proteins, genes of some small

RNAs• RNA polymerase III: genes encoding tRNAs, gene for 5S rRNA, genes

of some small RNAs

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3. MECHANISM OF TRANSCRIPTION:

Initiation: RNA polymerase binding to the promoter of gene which will be

translated.

Promotor: a sequence of DNA which indicates the point where the

transcription starts. Promoter is located before the first transcribed

nucleotide. It contains TATA box (formed mainly by T and A nucleotides).

RNA polymerase binding to promoter:

General transcription factors are involved in the binding. [FIG.]

Promotor is asymmetrical and it always binds RNA polymerase in one

direction while RNA polymerase can transcribe DNA template only in

3´→5´ direction.

→

Proper strand is only transcribed, i.e. template strand.

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3. MECHANISM OF TRANSCRIPTION:

Initiation: RNA polymerase binding to the promoter of gene which will be

translated.

Promotor: a sequence of DNA which indicates the point where the

transcription starts. Promoter is located before the first transcribed

nucleotide. It contains TATA box (formed mainly by T and A nucleotides).

RNA polymerase binding to promoter:

General transcription factors are involved in the binding. [FIG.]

Promotor is asymmetrical and it always binds RNA polymerase in one

direction while RNA polymerase can transcribe DNA template only in

3´→ 5´ direction.

→

Proper strand is only transcribed, i.e. template strand.

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After RNA polymerase binding to promoter:

RNA polymerase unwinds in front of it a short segment of DNA double

helix (accessibility of template DNA strand for transcription)

→

RNA polymerase adds to the first transcribed DNA nucleotide

complementary RNA nucleotide and thus the transcription is started.

Elongation phase of transcription:

RNA polymerase continues along template DNA strand, it unwinds

ahead a short segment of DNA double helix and at the same time it

synthesizes new RNA strand on the basis of complementary pairing

with the bases of template strand.

During transcription, a short hybrid double-stranded segment of DNA-RNA

is formed transiently. However, newly synthesized RNA strand is

released from binding to DNA soon.

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Termination of transcription:

Signal for the termination is a specific DNA sequence (terminator).

→

The release of RNA polymerase from binding to DNA and at the same

time the release of newly transcribed RNA strand.

Transcription machinery: 1 error/104 nucleotides

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4. POSTTRANSCRIPTIONAL PROCESSING:

Posttranscriptional RNA processing: modifications of RNA after its transcription

in the nucleus of eucaryotic cell and before its transport into cytoplasm and its

translation.

Modifications of both ends of transcribed RNA strand:• RNA capping: 7-methylquanosine is bound to the 5´end by the unusual

5´→5´ linkage via a bridge made of 3 phosphates (cap). [FIG.]• RNA polyadenylation: repeated adenine nucleotides (100-200) are bound to the 3

´end (poly-A end).

These two modifications increase the stability of mRNA.

RNA splicing: noncoding sequenses (introns) are removed from primary transcript

and codding sequenses (exons) are joined in given order. [FIG.] [FIG.]

Completed mRNA, which is ready for translation, is formed by the

posttranscriptional processing.

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4. POSTTRANSCRIPTIONAL PROCESSING:

Posttranscriptional RNA processing: modifications of RNA after its transcription

in the nucleus of eucaryotic cell and before its transport into cytoplasm and its

translation.

Modifications of both ends of transcribed RNA strand:• RNA capping: 7-methylquanosine is bound to the 5´end by the unusual

5´→5´ linkage via a bridge made of 3 phosphates (cap). [FIG.]• RNA polyadenylation: repeated adenine nucleotides (100-200) are bound to the 3

´end (poly-A end).

These two modifications increase the stability of mRNA.

RNA splicing: noncoding sequenses (introns) are removed from primary transcript

and codding sequenses (exons) are joined in given order. [FIG.] [FIG.]

Completed mRNA, which is ready for translation, is formed by the

posttranscriptional processing.

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Bacteriální gen

Kódující oblast

DNA3’5’

5’3’

Eukaryotní gen

Kódující oblast(exony)

Nekódující oblast(introny)

DNA3’5’

5’3’

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4. POSTTRANSCRIPTIONAL PROCESSING:

Posttranscriptional RNA processing: modifications of RNA after its transcription

in the nucleus of eucaryotic cell and before its transport into cytoplasm and its

translation.

Modifications of both ends of transcribed RNA strand:• RNA capping: 7-methylquanosine is bound to the 5´end by the unusual

5´→5´ linkage via a bridge made of 3 phosphates (cap). [FIG.]• RNA polyadenylation: repeated adenine nucleotides (100-200) are bound to the 3

´end (poly-A end).

These two modifications increase the stability of mRNA.

RNA splicing: noncoding sequenses (introns) are removed from primary transcript

and codding sequenses (exons) are joined in given order. [FIG.] [FIG.]

Completed mRNA, which is ready for translation, is formed by the

posttranscriptional processing.

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5. RNA SPLICING:

Introns are often longer than exons.

Exons (in an average gene): ~ 1000 nukleotides

Introns (in an average gene): 5000-20 000 nukleotides

Spliceosomes: large complexes of ribonucleoproteins and proteins

which carry out splicing.

Present ribonucleoproteins contains small nuclear RNAs (snRNAs).

Small nuclear RNAs recognize exon-intron boundaries and they form

small nuclear ribonucleoprotein particles (snRNPs) referred to as

„snurps“.

„Snurps“ form the core of spliceosome.

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Mechanism of RNA splicing:

at the place of relevant intron, a loop is formed due to the interaction of

spliceosome with RNA → ends of neighboring exons get closer → RNA

strand between intron and exon is interrupted and corresponding ends

of neighboring exons are joined → the loop of excised intron is

released and at the same time components of spliceosome are

released [FIG.]

Evolutionary significance of intron existence:• Possibility of alternative splicing (more proteins from one gene).• Increased probability of genetic recombination between exons of

different genes.

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Mechanism of RNA splicing:

at the place of relevant intron, a loop is formed due to the interaction of

spliceosome with RNA → ends of neighboring exons get closer → RNA

strand between intron and exon is interrupted and corresponding ends

of neighboring exons are joined → the loop of excised intron is

released and at the same time components of spliceosome are

released [FIG.]

Evolutionary significance of intron existence:• Possibility of alternative splicing (more proteins from one gene).• Increased probability of genetic recombination between exons of

different genes.

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6. ESSENCE OF TRANSLATION:

Translation: the synthesis of new polypeptide chain (protein) according

to genetic information saved in corresponding mRNA.

The sequence of amino acids is determined by the sequence of bases

in corresponding mRNA. It happens on the basis of genetic code.

It is a turn (translation) from the language of nucleotides into the

language of amino acids.

Translation apparatus reads mRNA in 5´→3´ direction.

New polypeptide chain is synthesized from the N end towards the C

end.

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Translation is carried out on ribosomes.

Together with mRNA (it is carrying genetic information), tRNA plays a

key role during translation.

Individual types of tRNA function as adaptors recognizing

corresponding codon for amino acid which they carry. [FIG.]

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7. STRUCTURE AND FUNCTION OF RIBOSOME:

Ribosome: a large complex which comprises several types of rRNA and a

big number of various proteins.

Eukaryotic ribosome (80S ribosome):• Small subunit: 1 type of rRNA (18S) & 33 proteins.• Large subunit: 3 types of rRNA (5S, 5.8S, 28S) & 49 proteins. [FIG.]

There are 4 specific binding sites on ribosome:• mRNA binding site

and three tRNA binding sites, i.e.• A site (aminoacyl-tRNA): it binds tRNA carrying relevant amino acid• P site (peptidyl-tRNA): it binds peptidyl-tRNA (peptide bound to tRNA)• E site (exit): tRNA is released from ribosome here [FIG.]

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7. STRUCTURE AND FUNCTION OF RIBOSOME:

Ribosome: a large complex which comprises several types of rRNA and a

big number of various proteins.

Eukaryotic ribosome (80S ribosome):• Small subunit: 1 type of rRNA (18S) & 33 proteins.• Large subunit: 3 types of rRNA (5S, 5.8S, 28S) & 49 proteins. [FIG.]

There are 4 specific binding sites on ribosome:• mRNA binding site

and three tRNA binding sites, i.e.• A site (aminoacyl-tRNA): it binds tRNA carrying relevant amino acid• P site (peptidyl-tRNA): it binds peptidyl-tRNA (peptide bound to tRNA)• E site (exit): tRNA is released from ribosome here [FIG.]

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8. STRUCTURE AND FUNCTION OF tRNA:

tRNA: about 80 nucleotides, it contains some minor bases and

nucleotides derived from them (pseudouridine - ψ, dihydrouridine - D).

Structure of tRNA: 4 short double-stranded segments (complementary pairing)

3 double-stranded segments are terminated by single-stranded loop

it has a character of clover leaf

Relevant amino acid is bound at the 3´end (terminal sequence of CCA)

by energy-rich bond.

Energy of this bond is used for the formation of peptide bond during

translation.

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Anticodon: sequence of 3 nucleotides of tRNA which is complementary to

the triplet of bases on mRNA encoding amino acid carried by this tRNA.

[FIG.]

Some of tRNAs require, for binding to mRNA during translation, precise

complementary pairing only on the first two positions of codon.

→

For amino acid encoded by more triplets with the same first two bases,

only one tRNA is sufficient for translation.

Thus only 31 types of tRNA are sufficient for 20 amino acids encoded by

61 codons.

Aminoacyl-tRNA synthetase: it performs binding of amino acid to relevant

tRNA with corresponding anticodon. Each amino acid has own specific

aminoacyl-tRNA synthetase.

These synthetases carry out decoding of the genetic code.

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Anticodon: sequence of 3 nucleotides of tRNA which is complementary to

the triplet of bases on mRNA encoding amino acid carried by this tRNA.

[FIG.]

Some of tRNAs require, for binding to mRNA during translation, precise

complementary pairing only on the first two positions of codon.

→

For amino acid encoded by more triplets with the same first two bases,

only one tRNA is sufficient for translation.

Thus only 31 types of tRNA are sufficient for 20 amino acids encoded by

61 codons.

Aminoacyl-tRNA synthetase: it performs binding of amino acid to relevant

tRNA with corresponding anticodon. Each amino acid has own specific

aminoacyl-tRNA synthetase.

These synthetases carry out decoding of the genetic code.

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9. MECHANISM OF TRANSLATION:

The translation of mRNA always starts by the codon AUG which encodes

methionine. → Every new polypeptide chain starts with methionine.

A specific initiator tRNA carrying methionine is required for the initiation of

translation.

Initiation of translation: binding of the initiator tRNA together with other

initiator factors to the small ribosomal subunit

→ complex binds to the 5´end of mRNA (it recognizes the cap)

→ it slides along mRNA in 5´→3´ direction until it recognizes the first AUG codon

→ the large subunit binds (ribosome is completed) while the initiator tRNA is bound at the P site

→ further aminoacyl-tRNA, with the anticodon corresponding to the second codon of mRNA after AUG, binds at the A site [FIG.]

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Elongation of translation: it starts by binding of further

aminoacyl-tRNA at the A site

→ polypeptide chain is released from tRNA at the P site and it is bound by peptide bond to the amino acid carried by the tRNA at the A site

→ at the same time tRNA carrying prolonged polypeptide chain is shifted from the A site to the P site and now free tRNA is shifted from the P site to E site

→ thus, the A site is freed for binding of further aminoacyl-tRNA

→ in the last step, tRNA is released from the E site and whole the cycle of elongation can be repeated [FIG.]

Peptidyl transferase: it catalyzes the formation of peptide bond, it is a

part of the large ribosomal subunit.

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Elongation of translation: it starts by binding of further

aminoacyl-tRNA at the A site

→ polypeptide chain is released from tRNA at the P site and it is bound by peptide bond to the amino acid carried by the tRNA at the A site

→ at the same time tRNA carrying prolonged polypeptide chain is shifted from the A site to the P site and now free tRNA is shifted from the P site to E site

→ thus, the A site is freed for binding of further aminoacyl-tRNA

→ in the last step, tRNA is released from the E site and whole the cycle of elongation can be repeated [FIG.]

Peptidyl transferase: it catalyzes the formation of peptide bond, it is a

part of the large ribosomal subunit.

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Termination of translation: it takes place when one of the three stop

codons (UAA, UAG, UGA) appears at the A site

→ stop codons are not recognized by any tRNA and protein release factors are bound instead of tRNA

→ a consequence is that peptidyl transferase catalyzes binding of water molecule instead of amino acid

→ polypeptide chain is ternated and it is released from ribosome[FIG.]

Polyribosomes: the initiation of translation happens repeatedly on

individual mRNA molecules → several ribosomes perform translation

on one mRNA molecule at the same time [FIG.]

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Termination of translation: it takes place when one of the three stop

codons (UAA, UAG, UGA) appears at the A site

→ stop codons are not recognized by any tRNA and protein release factors are bound instead of tRNA

→ a consequence is that peptidyl transferase catalyzes binding of water molecule instead of amino acid

→ polypeptide chain is ternated and it is released from ribosome[FIG.]

Polyribosomes: the initiation of translation happens repeatedly on

individual mRNA molecules → several ribosomes perform translation

on one mRNA molecule at the same time [FIG.]

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LITERATURE:

• Alberts B. et al.: Essential Cell Biology. Garland Science. New York and London, pp. 231 260, 2010