Transcript 3D-structure of bacterial ribosomes, the machines that make

3D-structure of bacterial ribosomes, the machines that make proteins
Components required for protein-synthesis in E. coli
The content of RNA and protein chains in E. coli ribosomes
Possible folding structures of 16S og 5S rRNA
The difference between bacterial and eukaryotic ribosomes
The general structure of tRNA, the transportes of amino acids to the ribosomes
3D-structure of tRNAPhe from yeast
FIGURE 2.16
Structure of tRNA Allows Wobble in the Third Position
Transfer RNA recognizes the codons along mRNA and presents the correct amino acid for each
codon. The first position of the anticodon on tRNA matches the third position of the codon.
Biotechnology by Clark and Pazdernik
Copyright © 2012 by Academic Press. All rights reserved.
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The information in mRNA is translated to amino acid sequences (protein) via a
three-letter system called the genetic code
FIGURE 2.15
The Genetic Code
The 64 codons found in mRNA are shown with their corresponding amino acids. As usual, bases are
read from 5’ to 3’ so that the first base is at the 5’ end of the codon. Three codons (UAA, UAG, UGA)
have no cognate amino acid but signal stop. AUG (encoding methionine) and, much less often, GUG
(encoding valine) act as start codons. To locate a codon, find the first base in the vertical column on
the left, the second base in the horizontal row at the top, and the third base in the vertical column on
the right.
Biotechnology by Clark and Pazdernik
Copyright © 2012 by Academic Press. All rights reserved.
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Transcription and translation are coupled in prokaryotes, but not in eukaryotes
The N-terminal ends of proteins are made first
Aminoacylation of tRNA by aminoacyl-tRNA synthetase
General structure of amino-acyl tRNA
The initiating amino acid at the start codon is N-formylmethionine
FIGURE 2.17
Translation in Prokaryotes
(A) Initiation of translation begins with the association of the small ribosome subunit with the Shine-Dalgarno sequence
(S-D sequence) on the mRNA. Next, the initiator tRNA that reads AUG is charged with fMet. The charged initiator tRNA
associates with the small ribosome subunit and finds the start codon. Assembly is helped by initiation factors (IF1, IF2,
and IF3)—not shown. (B) During elongation peptide bonds are formed between the amino acids at the A-site and the Psite. The movement of the ribosome along the mRNA and addition of a new tRNA to the A-site are controlled by
elongation factors (also not shown). (C) Termination requires release factors. The various components dissociate. The
completed protein folds into its proper three-dimensional shape.
Biotechnology by Clark and Pazdernik
Copyright © 2012 by Academic Press. All rights reserved.
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FIGURE 2.18
Translation in Eukaryotes
(A) Assembly of the small subunit plus initiator Met-tRNA involves the binding of factors eIF3 and eIF2. (B) The cap
binding protein of eIF4 attaches to the mRNA before it joins the small subunit. (C) The mRNA binds to the small subunit
via cap binding protein and the 40S initiation complex is assembled. (D) Assembly of the large subunit requires factor
eIF5. After assembly, eIF2 and eIF3 depart.
Biotechnology by Clark and Pazdernik
Copyright © 2012 by Academic Press. All rights reserved.
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Formation of the initiation
complex in bacteria
The Shine-Dalgarno sequence in mRNA interacts with 16 rRNA in the ribosome
De viktigste punktene i proteinsyntesen i bakterier:
•Den første aminosyra er alltid formylmetionin, uavhengig av hva startkodonet
(nesten alltid AUG eller GUG) er.
•Før peptidbindinger lages må aminosyrene aktiveres ved å kondensere med ATP.
Denne reaksjonen drives av spalting av en fosfodiesterbinding i ATP slik at PP frigjøres.
Etter dette kobles aminosyren på sitt aktuelle tRNA ved hjelp av aminoacyl tRNA
syntetase.
•30S subenheten av ribosomet bindes til mRNA slik at AUG blir posisjonert i P-setet
(peptidylsetet). Husk at det er her Shine-Dalgarno sekvensen i mRNA kommer inn ved å
basepare med en del av 16S rRNA.
•tRNA med formylmetionin rekrutteres til P-setet ved binding til AUG i mRNA.
•50S subenheten rekrutteres til komplekset.
•Neste aminosyre rekrutteres via sitt tRNA til kodon 2 i mRNA ved A-setet (aminoacylsetet) i ribosomet.
•En peptidbinding dannes ved at formylmetionin linkes til aminosyren i A-setet.
•tRNA med dipeptidet translokeres fra A-setet til P-setet.
•En tredje aminosyre rekrutteres via sitt tRNA til kodon 3 i mRNA ved A-setet. Ny
peptidpinding dannes som over.
•Prosessen fortsetter til stopp-kodonet. Dette senses av release faktor og proteinet
forlater ribosomet i Exit site. Ribosomet spaltes i 30S og 50S subenheter igjen.
•Det inngår mange proteinfaktorer (initieringsfaktorer, elongeringsfaktorer etc) i de ulike
stegene, men dere trenger ikke å huske navnene på disse eller hvor de inngår. Derimot
er det svært viktig å være klar over at det forbrukes veldig mye GTP. Siden det lages
veldig mye proteiner i levende celler blir dermed proteinsyntesen en energimessig sett
veldig kostbar prosess.
Proteins and folding and posttranslatoric modification
Proteins can be denatured, i.e by heating. This means that the folding structure
collapses, resulting in loss of functionality
Proteins from thermophilic organisms can resist boiling
Some proteins fold correctly by themselves after retransfer to low temperatures, but
most proteins don’t
During production many proteins can only obtain correct folding if assisted by other
proteins (chaperones) during production in living cells
If large quantities of a specific chaperone-dependent protein is produced in a cell, it may
become misfolded. This represents a very serious problem in biotechnology
If a protein is misfolded during production it may in some, but not all cases, be correctly
refolded in the laboratory
Many proteins, particularly from eukaryotes, are further modified in the cells after
translation. This can involve phosphorylation, glycosylation etc. The modifications have
various biolological functions, and this is also very important in biotechnology.