Initiation of translation in prokaryotes: initiation factors, initiator codons, 3'end of RNA small ribosomal subunit

Июль 24, 2022

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Initiation of translation in prokaryotes: initiation factors, initiator codons, 3'end of RNA small ribosomal subunit

Содержание

2. In molecular biology and genetic, translation is the process in which ribosomes in a cell's cytoplasm
3. Translation proceeds in three phases: Initiation: The ribosome assembles around the target mRNA. The first tRNA
4. In bacteria, translation occurs in the cytoplasm, where the large and small subunits of the ribosome
5. A number of antibiotics act by inhibiting translation. These include anisomycin, cycloheximide, chloramphenicol, tetracycline, streptomycin, erythromycin,
6. Translation initiation: Initiation factors Prokaryotes require the use of three initiation factors: IF1, IF2, and IF3,
7. Translation initiation: Initiation factors IF2 binds to an initiator tRNA and controls the entry of tRNA
8. Translation initiation: Initiation factors IF3 is not universally found in all bacterial species but in E.
10. The fact that cells typically contain many ribosomes reflects the central importance of protein synthesis in
11. * Each ribosome contains one copy of the rRNAs and one copy of each of the
12. A noteworthy feature of ribosomes is that they can be formed in vitro by self-assembly of
13. Ribosome: rRNA -rRNAs are much more than structural components of ribosome directly responsible for the key
14. Ribosome: 16S rRNA 1)A site: binding site for aminoacyl-tRNA 2)P site: binding site for peptidyl-tRNA 3)E
15. Ribosome: 16S rRNA (Zwieb & Brimacombe I979)
16. Ribosome: 16S rRNA The arrangement of the 16S rRNA creates a 5' domain, central domain, 3'
17. Ribosome: 16S rRNA The 3' major domain contains 15 helical elements and composes the head. The
19. First, the small subunit associates with the mRNAby base-pairing interaction between RBS and 16S rRNA -the
20. Ribosome: 3’ end of 16 s rRNA Unique localization of the 3' end of the RNA
21. Ribosome: 3’ end of 16 s rRNA Cross-linking studies have shown that the nucleic acid-binding domain
22. Antibiotics affecting 16S rRNA Colicin E3 (protein antibiotic from E.coli) makes a single cut in the
23. Shine-Dalgarno sequence
24. The Shine-Dalgarno (SD) sequence is a ribosomal binding site in bacterial and archaeal messenger RNA, generally
27. several examples of the Shine-Dalgarno sequence
28. REFERENCE The Cell: A Molecular Approach. 2nd edition.Cooper GM. Sunderland (MA): Sinauer Associates; 2000. Watson J
30. Скачать презентацию

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In molecular biology and genetic, translation is the process in which ribosomes in a cell's cytoplasm create
proteins, following transcrip-tion of DNA to RNA in

the cell's nucleus. The entire process is a part of gene expression.

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Translation proceeds in three phases:
Initiation: The ribosome assembles around the target

mRNA. The first tRNA is attached at the start codon.
Elongation: The tRNA transfers an amino acid to the tRNA corresponding to the next codon. The ribosome then moves (translocates) to the next mRNA codon to continue the process, creating an amino acid chain.
Termination: When a stop codon is reached, the ribosome releases the polypeptide.

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In bacteria, translation occurs in the cytoplasm, where the large and

small subunits of the ribosome bind to the mRNA. In eukaryotes, translation occurs in the cytosol or across the membrane of the endoplasmic reticulum in a process called vectorial synthesis. In many instances, the entire ribosome/mRNA complex binds to the outer membrane of the rough endoplasmic reticulum (ER); the newly created polypeptide is stored inside the ER for later vesicle transport and secretion outside of the cell.
Many types of transcribed RNA, such as transfer RNA, ribosomal RNA, and small nuclear RNA, do not undergo translation into proteins.

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A number of antibiotics act by inhibiting translation. These include anisomycin,

cycloheximide, chloramphenicol, tetracycline, streptomycin, erythromycin, and puromycin. Prokaryotic ribosomes have a different structure from that of eukaryotic ribosomes, and thus antibiotics can specifically target bacterial infections without any harm to a eukaryotic host's cells.

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Translation initiation: Initiation factors
Prokaryotes require the use of three initiation factors:

IF1, IF2, and IF3, for translation.
IF1 associates with the 30S ribosomal subunit in the A site and prevents an aminoacyl-tRNA from entering. It modulates IF2 binding to the ribosome by increasing its affinity. It may also prevent the 50S subunit from binding, stopping the formation of the 70S subunit. It also contains a β-domain fold common for nucleic acid binding proteins.

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Translation initiation: Initiation factors
IF2 binds to an initiator tRNA and controls

the entry of tRNA onto the ribosome. IF2, bound to GTP, binds to the 30S P site. After associating with the 30S subunit, fMet-tRNAf binds to the IF2, then IF2 transfers the tRNA into the partial P site. When the 50S subunit joins, it hydrolyzes GTP to GDP and Pi, causing a conformational change in the IF2 that causes IF2 to release and allow the 70S subunit to form.

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Translation initiation: Initiation factors
IF3 is not universally found in all bacterial

species but in E. coli it is required for the 30S subunit to bind to the initiation site in mRNA. In addition, it has several other jobs including the stabilization of free 30S subunits, enables 30S subunits to bind to mRNA and checks for accuracy against the first aminoacyl-tRNA. It also allows for rapid codon-anticodon pairing for the initiator tRNA to bind quickly to. IF3 is required by the small subunit to form initiation complexes, but has to be released to allow the 50S subunit to bind.

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The fact that cells typically contain many ribosomes reflects the central

importance of protein synthesis in cell metabolism. E. coli, for example, contain about 20,000 ribosomes, which account for approximately 25% of the dry weight of the cell, and rapidly growing mammalian cells contain about 10 million ribosomes.

Ribosome

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* Each ribosome contains one copy of the rRNAs and one

copy of each of the ribosomal proteins, with one exception: One protein of the 50S subunit is present in four copies.

Ribosome: Structure

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A noteworthy feature of ribosomes is that they can be formed in vitro by self-assembly

of their RNA and protein constituents. As first described in 1968 by Masayasu Nomura, purified ribosomal proteins and rRNAs can be mixed together and, under appropriate conditions, will reform a functional ribosome.
Initially, rRNAs were thought to play a structural role, providing a scaffold upon which ribosomal proteins assemble. However, with the discovery of the catalytic activity of other RNA molecules, the possible catalytic role of rRNA became widely considered. Consistent with this hypothesis, rRNAs were found to be absolutely required for the in vitro assembly of functional ribosomes.

Ribosome: rRNA

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Ribosome: rRNA
-rRNAs are much more than structural components of ribosome directly
responsible

for the key functions of the ribosome
*peptidyl transferase center is composed almost entirely of RNA
* 16S rRNA of small subunit is responsible for mRNA binding ;
*also function in the small subunit: anticodon loop and codon of mRNA
contact 16S rRNA;
-most ribosomal
proteins are in
periphery
*some proteins in
core for
stabilization
reasons

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Ribosome: 16S rRNA
1)A site: binding site for aminoacyl-tRNA
2)P site: binding

site for peptidyl-tRNA
3)E (denote exit) site: binding site for tRNA released after growing polypeptide chain has been transferred to the aminoacyl-tRNA (i.e., free tRNA)

Affinity label for the tRNA binding sites on the E. coli ribosome allowed the identification of A and P site proteins most likely associated with the peptidyl-transferase activity; labelled proteins are L27, L14, L15, L16, L2;

Additional research has demonstrated that the S1 and S21 proteins, in association with the 3'-end of 16S ribosomal RNA, are involved in the initiation of translation.

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Ribosome: 16S rRNA
(Zwieb & Brimacombe I979)

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Ribosome: 16S rRNA
The arrangement of the 16S rRNA creates a 5'

domain, central domain, 3' major domain, and a 3' minor domain.

The 5' domain consists of 19 double helices that makes up the bulk of the body.

The central domain of the rRNA generates the platform and is an elongated, curved structure of nine helices, with the junction of helices 20, 21, and 22 being at the heart of it.

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Ribosome: 16S rRNA
The 3' major domain contains 15 helical elements and

composes the head.

The 3' minor domain contains 2 helices and projects from the subunit to interact with the 50S subunit.

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First, the small subunit associates with the mRNAby base-pairing interaction between

RBS and 16S rRNA
-the small subunit is positioned on
mRNA such that the start
codon will be in the P site
when the large subunit
joins the complex

Ribosome: 3’ end of 16S rRNA

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Ribosome: 3’ end of 16 s rRNA
Unique localization of the 3'

end of the RNA on the upper portion of the subunit platform.. Ribosomal proteins SI and S21, which have been cross-linked to the 3' end of the RNA, are localized near each other and near the end of the platform. Initiation factor IF3, to which the oxidized 3' end of the RNA has also been linked, has been itself cross-linked to ribosomal proteins S1, Sll, S12, S13, S19, and S21; antigenic determinants of each of these proteins have been localized either on the subunit platform or on nearby parts of the upper portion of the subunit

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Ribosome: 3’ end of 16 s rRNA
Cross-linking studies have shown that

the nucleic acid-binding domain of S1 is aligned with a region of the mRNA upstream of the SD, suggesting that S1 may interact with 5' parts of the Translation initiation region. Consistent with this observation, A/U-rich sequences in front of the SD or downstream of the initiator codon enhance protein synthesis. Disruption of the E. coli gene coding for S1 has been reported to be lethal.

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Antibiotics affecting 16S rRNA
Colicin E3 (protein antibiotic from E.coli) makes a
single

cut in the 16S rRNA of 70S ribosomes, these include the loss of activity.
Pactamycin (Pct) was isolated from Streptomyces pactum as a potential new human antitumor drug, but in fact a potent inhibitor of translation in all three kingdoms, eukarya, bacteria, and archaea (Bhuyan et al., 1961; Mankin, 1997). For this reason, the drug is expected to interact with highly conserved regions of 16S RNA.
Streptomycin and spectinomycin are typical examples which function by binding to specific sites on prokaryotic rRNA and affecting the fidelity of protein synthesis. Binding of drug to the 16S subunit near the A-site of the 30S subunit leads to a decrease in translational accuracy and inhibition of the translocation of the ribosome.

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Shine-Dalgarno sequence

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The Shine-Dalgarno (SD) sequence is a ribosomal binding site in bacterial and archaeal messenger RNA, generally

located around 8 bases upstream of the start codon AUG.

The RNA sequence helps recruit the ribosome to the messenger RNA (mRNA) to initiate protein synthesis by aligning the ribosome with the start codon.

The Shine-Dalgarno sequence was proposed by Australian scientists John Shine and Lynn Dalgarno

The Shine-Dalgarno sequence exists both in bacteria and archaea. It is also present in some chloroplast and mitochondrial transcripts.

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several examples of the Shine-Dalgarno sequence

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REFERENCE
The Cell: A Molecular Approach. 2nd edition.Cooper GM. Sunderland (MA): Sinauer Associates;

2000.
Watson J D, Baker T A , Bell S P, Gann A, Levine M, Losick R. Molecular Biology of the Gene. 5th edition. Pearson education 2004.
J. E. KREBS, E.S. GOLDSTEIN, S.T. KILPATRICK. Lewin’s genes XI. Copyright© 2 0 1 4 by Jones & B artlett Learning, LLC, an Ascend Learning Company
Daniel H. Lackner et al., Translational Control of Gene Expression: From Transcripts to Transcriptomes, International Review of Cell and Molecular Biology, 2008, 271, 200-238.
The Cell: A Molecular Approach. 2nd edition.Cooper GM. Sunderland (MA): Sinauer Associates; 2000.
Czernilofsky, A; Kurland, C.G.; Stöffler, G. (1975). "30S RIBOSOMAL-PROTEINS ASSOCIATED WITH 3'-TERMINUS OF 16S RNA". FEBS Letters. ELSEVIER SCIENCE BV. 58 (1): 281–284.
H. G. WITTMANN . Structure and evolution of ribosomes . Proc. R. Soc. Lond. B 216, 117-135 (1982)
Zwieb, C. & Brimacombe, R. I979 Nucl. Acids Res. 6, 1775-1790.
HELEN MCKUSKIE OLSON* AND DOHN G. GLITZ. Ribosome structure: Localization of 3' end of RNA in small subunit by immunoelectronmicroscopy. Proc. Natl. Acad. Sci. USA. Vol. 76, No. 8, pp. 3769-3773, August 1979
Heinz-Giinter WITTMANN. Structure, Function and Evolution of Ribosomes. Eur. J. Biochem. 61, 1 - 13 (1976)

Initiation of translation in prokaryotes: initiation factors, initiator codons, 3'end of RNA small ribosomal subunit

Содержание

In molecular biology and genetic, translation is the process in which ribosomes in a cell's cytoplasm create proteins, following transcrip-tion of DNA to RNA in

Translation proceeds in three phases: Initiation: The ribosome assembles around the target

In bacteria, translation occurs in the cytoplasm, where the large and

A number of antibiotics act by inhibiting translation. These include anisomycin,

Translation initiation: Initiation factors Prokaryotes require the use of three initiation factors:

Translation initiation: Initiation factors IF2 binds to an initiator tRNA and controls

Translation initiation: Initiation factors IF3 is not universally found in all bacterial

The fact that cells typically contain many ribosomes reflects the central

* Each ribosome contains one copy of the rRNAs and one

A noteworthy feature of ribosomes is that they can be formed in vitro by self-assembly

Ribosome: rRNA-rRNAs are much more than structural components of ribosome directlyresponsible

Ribosome: 16S rRNA1)A site: binding site for aminoacyl-tRNA 2)P site: binding

Ribosome: 16S rRNA(Zwieb & Brimacombe I979)

Ribosome: 16S rRNA The arrangement of the 16S rRNA creates a 5'

Ribosome: 16S rRNAThe 3' major domain contains 15 helical elements and