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@@ -1,10 +0,0 @@
|
||||
---
|
||||
tags:
|
||||
- biokemi
|
||||
- translation
|
||||
- anteckningar
|
||||
föreläsare: Ana Luis
|
||||
url: https://www.youtube.com/watch?v=gjjp-NUaCXE
|
||||
date: 2024-11-25
|
||||
title: Translationsanteckningar
|
||||
---
|
||||
@@ -1,53 +0,0 @@
|
||||
---
|
||||
tags:
|
||||
- biokemi
|
||||
- translation
|
||||
- instuderingsuppgifter
|
||||
föreläsare: Ana Luis
|
||||
---
|
||||
|
||||
## Genetic code
|
||||
#### Describe the main features of the genetic code.
|
||||
|
||||
#### What is degeneracy of the genetic code and what its biological significance?
|
||||
|
||||
## Translation and tRNA
|
||||
#### What is translation?
|
||||
|
||||
#### What is a tRNA and what is its function in protein synthesis?
|
||||
|
||||
#### What are the general characteristics of a tRNA?
|
||||
|
||||
#### Why the 3’ CCA terminal region in a tRNA is also know as the acceptor arm?
|
||||
|
||||
#### What is the wobble effect and which base in the anticodon determines the wobble effect?
|
||||
|
||||
#### Explain why aminoacyl-tRNA synthetases are the ‘true reads’ of the genetic code?
|
||||
|
||||
#### How do aminoacyl-tRNA synthetases work? Describe active and editing sites.
|
||||
|
||||
## Ribosomes
|
||||
#### What is a ribosome and what are its different components?
|
||||
|
||||
#### Which components of the ribosome are critical to its structure and function?
|
||||
|
||||
#### Describe the three binding sites (A, P, and E) and which tRNAs are found in each site.
|
||||
#### What are the differences between bacterial and eukaryotic ribosomes?
|
||||
|
||||
#### How can the ribosome be used as a structure to development of new antibiotics?
|
||||
|
||||
## Protein translation mechanism
|
||||
#### What are the steps of protein translation?
|
||||
|
||||
#### What are the characteristics of the initiation region in bacteria?
|
||||
|
||||
#### Explain why the reading frame is establish during the initiation step of protein synthesis?
|
||||
#### How does protein initiation start and what role initiation factors play?
|
||||
#### What is the role of elongation and translocation factors?
|
||||
#### What is the role of release factors in protein synthesis?
|
||||
|
||||
#### What is a polysome and what is its biological significance?
|
||||
|
||||
#### What are the differences between bacteria and eukaryotic protein biosynthesis?
|
||||
|
||||
#### Streptomycin is a bacterial antibiotic that blocks protein biosynthesis. Describe how this antibiotic works and which step of protein biosynthesis is inhibited.
|
||||
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---
|
||||
tags:
|
||||
- biokemi
|
||||
- translation
|
||||
- lärandemål
|
||||
föreläsare: Ana Luis
|
||||
date: 2025-11-25
|
||||
---
|
||||
|
||||
- tRNA stuktur och funktion.
|
||||
- Aminoacylering av tRNA.
|
||||
- Ribosomens struktur och funktion.
|
||||
- Mekanismen för proteinsyntes (initiering, elongering, translokation och terminering).
|
||||
- Regleringsmekanismer för proteinsyntesen
|
||||
|
||||
Beskriva överföringen av genetisk information från mRNA till protein.
|
||||
@@ -1,568 +0,0 @@
|
||||
---
|
||||
tags:
|
||||
- biokemi
|
||||
- translation
|
||||
- slides
|
||||
föreläsare: Ana Luis
|
||||
---
|
||||
|
||||
Chapter 8 and Chapter 20
|
||||
Ana Luis
|
||||
ana.luis@medkem.gu.se
|
||||
2025/04/28
|
||||
|
||||
---
|
||||
|
||||
Protein Biosynthesis
|
||||
|
||||
(Translation)
|
||||
Chapter 8 and Chapter 20
|
||||
Ana Luis
|
||||
ana.luis@medkem.gu.se
|
||||
2025/04/28
|
||||
|
||||
----
|
||||
|
||||
Learning goals
|
||||
|
||||
• Explain how nucleic acid information is translated into an amino acid sequence and define the role of aminoacyl tRNA synthetases in this process
|
||||
• Protein biosynthesis requires the translation of nucleotide sequences into amino acid sequences
|
||||
• Describe features of the genetic code
|
||||
• tRNA structure and function
|
||||
• Aminoacyl-tRNA synthetases establish the genetic code
|
||||
|
||||
• Define the role of ribosomes in protein synthesis
|
||||
• Structure and function of ribosomes
|
||||
• Mechanism of protein synthesis (initiation, elongation, translocation and termination)
|
||||
|
||||
• Describe how certain chemicals can inhibit protein synthesis
|
||||
• Antibiotics and toxins inhibit protein synthesis
|
||||
|
||||
----
|
||||
|
||||
Why is important to understand the translation mechanisms?
|
||||
![[Pasted image 20251125093128.png]]
|
||||
### TRANSLATION
|
||||
|
||||
#### Inhibition of translation of specific mRNAs can lead to diseasess
|
||||
- Fragile X mental retardation syndrome:
|
||||
- absence of the set of protein isoforms, derived from
|
||||
alternative splicing of the Fragile X mental retardation
|
||||
gene 1 (FMR1)
|
||||
|
||||
#### Bacterial toxins can block of protein biosynthesis protein biosynthesis
|
||||
- Diphtheria toxin
|
||||
|
||||
#### Several antibiotics are inhibitors
|
||||
- Streptomycin
|
||||
- Tetracycline
|
||||
- …..
|
||||
|
||||
⸻
|
||||
|
||||
Translation = the process of protein biosynthesis
|
||||
|
||||
Adenine (A)
|
||||
Cytosine (C)
|
||||
Guanine (G)
|
||||
Uracil (U)
|
||||
Nucleic acid
|
||||
|
||||
→
|
||||
|
||||
Amino acids
|
||||
(A) Alanine
|
||||
(R) Arginine
|
||||
(N) Asparagine
|
||||
(D) Aspartic acid
|
||||
(C) Cysteine
|
||||
(E) Glutamic acid
|
||||
(Q) Glutamine
|
||||
(G) Glycine
|
||||
(H) Histidine
|
||||
(I) Isoleucine
|
||||
(L) Leucine
|
||||
(K) Lysine
|
||||
(M) Methionine
|
||||
(F) Phenylalanine
|
||||
(P) Proline
|
||||
(S) Serine
|
||||
(T) Threonine
|
||||
(W) Tryptophan
|
||||
(Y) Tyrosine
|
||||
(V) Valine
|
||||
|
||||
-----
|
||||
|
||||
Translation = the process of protein biosynthesis
|
||||
|
||||
The basics of protein biosynthesis are the same across all kingdoms of life:
|
||||
• mRNA is decoded in the 5′-to-3′ direction one codon at a time
|
||||
• the protein is synthesized in the amino-to-carboxyl direction
|
||||
mRNA
|
||||
A U G G U G G C U A A G C G G U G A
|
||||
Methionine Valine Alanine Lisine Arginine Stop
|
||||
5’ 3’
|
||||
Amino group
|
||||
Carboxyl group
|
||||
Amino group
|
||||
Carboxyl
|
||||
|
||||
⸻
|
||||
|
||||
tRNA
|
||||
|
||||
a.a.
|
||||
Translation = the process of protein biosynthesis
|
||||
• anticodon - portion of the tRNA that base pairs with the codon
|
||||
C G I
|
||||
3’ 5’
|
||||
G C C
|
||||
5’ 3’
|
||||
Codon
|
||||
• codon - three coding bases on the mRNA template
|
||||
• transfer RNA (tRNA) - function as adaptor molecules between a codon and
|
||||
an amino acid (a.a.)
|
||||
Anticodon
|
||||
mRNA
|
||||
The basics of protein biosynthesis are the same across all kingdoms of life:
|
||||
The correct protein biosynthesis requires an accurate recognition of codons by anticodons
|
||||
|
||||
-----
|
||||
|
||||
General characteristics of Transfer RNA (tRNA) molecules
|
||||
|
||||
• Each tRNA is a single chain containing between 73 and 93 nucleotides.
|
||||
• The secondary structure resembles a cloverleaf
|
||||
(~50% of the nucleotides are base-paired)
|
||||
Five groups of bases are not base-paired, but participate in
|
||||
hydrogen-bonding interactions:
|
||||
• 3′ CCA terminal region (acceptor stem)
|
||||
• TψC loop
|
||||
• “extra arm”
|
||||
• anticodon loop
|
||||
• DHU loop
|
||||
• The three-dimensional structure is L-shaped
|
||||
• tRNAs contain 7 to 15 unusual bases
|
||||
• methylated or demethylated derivatives
|
||||
of A, U, C, and G
|
||||
|
||||
⸻
|
||||
|
||||
• At the 3’ end, an activated amino acid is attached to a hydroxyl group of
|
||||
adenosine in the CCA region of the acceptor stem
|
||||
• the CCA region has the ability to change its conformation during protein synthesis
|
||||
• The anticodon loop is near the center of the sequence
|
||||
General characteristics of tRNA molecules
|
||||
|
||||
⸻
|
||||
|
||||
Genetic code
|
||||
|
||||
C G I
|
||||
3’ 5’
|
||||
G C C
|
||||
5’ 3’
|
||||
Codon
|
||||
Anticodon
|
||||
mRNA
|
||||
• Genetic code: the relation between the sequence of bases in DNA
|
||||
and the sequence of amino acids in proteins
|
||||
mRNA
|
||||
A U G G U G G C U A A G C G G U G A
|
||||
Methionine Valine Alanine Lysine Arginine Stop
|
||||
5’ 3’
|
||||
DNA
|
||||
PROTEIN
|
||||
|
||||
⸻
|
||||
|
||||
Amino acids are encoded by groups of three bases starting from a fixed point
|
||||
|
||||
• Features of the Genetic code:
|
||||
– three nucleotides (codon) encode an amino acid
|
||||
– has directionality
|
||||
– nonoverlapping
|
||||
– has no punctuation
|
||||
– is degenerate (most amino acids are encoded by more
|
||||
than one codon)
|
||||
mRNA
|
||||
A U G G U G G C U A A G C G G U G A
|
||||
Methionine Valine Alanine Lysine Arginine Stop
|
||||
5’ 3’
|
||||
• 61 codons encode specify amino acids (20 in total)
|
||||
• 3 codons are stop codons that designate termination
|
||||
of translation.
|
||||
|
||||
⸻
|
||||
|
||||
’Wobble’ effect in base-pairing
|
||||
|
||||
’Wobble’ effect: Some tRNA molecules can recognize more than one codon
|
||||
tRNA a.a.
|
||||
1
|
||||
C G I
|
||||
3’ 5’
|
||||
G C C
|
||||
5’ 3’
|
||||
Codon
|
||||
Anticodon
|
||||
mRNA
|
||||
tRNA a.a.
|
||||
1
|
||||
C G I
|
||||
3’ 5’
|
||||
G C A
|
||||
5’ 3’
|
||||
Codon
|
||||
Anticodon
|
||||
mRNA
|
||||
|
||||
⸻
|
||||
|
||||
’Wobble’ effect in base-pairing
|
||||
|
||||
Anticodons base-pair with codons
|
||||
’Wobble’ effect: Some tRNA molecules can recognize more than one codon
|
||||
Codons that differ in either of their first two bases (from 5’) must be recognized by different tRNAs.
|
||||
The first base of the anticodon (5’) determines the degree of wobble
|
||||
• The redundancy, or degeneracy, of the genetic code indicates that recognition of
|
||||
the third base of a codon is sometimes less discriminating than the other two
|
||||
“wobble” = steric freedom in the pairing of the first base of the anticodon with the third base of the codon
|
||||
|
||||
⸻
|
||||
|
||||
’Wobble’ effect in base-pairing
|
||||
|
||||
“wobble hypothesis”: established hypothesis that predicts the binding of anticodons to codons
|
||||
TABLE 30.2 Allowed pairings at the third base of the
|
||||
codon according to the wobble hypothesis
|
||||
Anticodons base-pair with codons
|
||||
C
|
||||
C
|
||||
G
|
||||
|
||||
⸻
|
||||
|
||||
’Wobble’ effect in base-pairing
|
||||
|
||||
“wobble hypothesis”: established hypothesis that predicts the binding of anticodons to codons
|
||||
TABLE 30.2 Allowed pairings at the third base of the
|
||||
codon according to the wobble hypothesis
|
||||
C
|
||||
U
|
||||
A/
|
||||
G
|
||||
|
||||
⸻
|
||||
|
||||
’Wobble’ effect in base-pairing
|
||||
|
||||
“wobble hypothesis”: established hypothesis that predicts the binding of anticodons to codons
|
||||
TABLE 30.2 Allowed pairings at the third base of the codon
|
||||
Example: If first base of the anticodon is inosine, the
|
||||
anticodon can recognize three different codons
|
||||
Inosine is formed by the deamination of adenosine
|
||||
• has a heterocyclic nitrogen base that can form
|
||||
hydrogen bonds with adenine, cytosine and uracil
|
||||
The purine base inosine pairs with cytidine, uridine or adenosine
|
||||
|
||||
⸻
|
||||
|
||||
Amino acids required for protein biosynthesis must first be attached to specific tRNA molecules
|
||||
|
||||
• The attachment of a given amino acid to a particular tRNA establishes the genetic code
|
||||
Aminoacyl-tRNA synthetases
|
||||
Aminoacyl-tRNA synthetases attach specific amino acids to tRNAs
|
||||
tRNA
|
||||
amino acid
|
||||
Aminoacyl-tRNA
|
||||
amino
|
||||
• acid
|
||||
|
||||
⸻
|
||||
|
||||
Ester linkages couple amino acids to tRNA
|
||||
|
||||
The process of attaching an amino acid to tRNA is called aminosylation
|
||||
CCA arm of tRNA
|
||||
• Amino acids are bound to the 3’end of the tRNA via an ester bond
|
||||
between the carboxyl group on the amino acid and either the 2’ or 3’
|
||||
hydroxyl group of the terminal adenosine of the tRNA
|
||||
3’
|
||||
Aminoacyl-tRNA: Amino acid bound to tRNA
|
||||
3’
|
||||
|
||||
⸻
|
||||
|
||||
Each aminoacyl-tRNA synthetase is specific for a given amino acid
|
||||
|
||||
How aminoacyl-tRNA synthetases evolve to differentiate between different amino acids?
|
||||
A closer look at the amino acid threonine, valine and serine
|
||||
|
||||
⸻
|
||||
|
||||
Threonyl-tRNA Synthetase contains an activation site
|
||||
|
||||
Each aminoacyl-tRNA synthetase is specific for a given amino acid
|
||||
How aminoacyl-tRNA synthetases evolve to differentiate Threonine, Valine and Serine?
|
||||
Activation site
|
||||
responsible for activating threonine by binding it
|
||||
to adenosine triphospate (ATP) and further
|
||||
transfer of these amino acid to the tRNA molecule
|
||||
• Aminoacyl-tRNA synthetases have highly
|
||||
discriminating amino acid activation sites
|
||||
|
||||
⸻
|
||||
|
||||
Each aminoacyl-tRNA synthetase is specific for a given amino acid
|
||||
|
||||
• To avoid coupling to the incorrect amino acid, threonyl-tRNA synthetase (tRNAThr)
|
||||
contains a zinc ion at the active site that binds to the amino and hydroxyl groups
|
||||
of threonine
|
||||
• Valine is similar in overall structure to threonine but
|
||||
lacks the hydroxyl group, so it does not bind to tRNAThr
|
||||
• Serine is occasionally linked to tRNAThr because
|
||||
of the presence of the hydroxyl group
|
||||
(Thr)
|
||||
(Val)
|
||||
(Ser)
|
||||
How aminoacyl-tRNA synthetases evolve to differentiate Threonine, Valine and Serine?
|
||||
|
||||
⸻
|
||||
|
||||
Threonyl-tRNA Synthetase contains an activation site and an editing site
|
||||
|
||||
Activation site
|
||||
responsible for activating threonine by binding it
|
||||
to adenosine triphospate (ATP) and further
|
||||
transfer of these amino acid to the tRNA molecule
|
||||
Editing site:
|
||||
acts as a profreader and removes any incorrect
|
||||
bound amino acid from the tRNA molecule
|
||||
|
||||
⸻
|
||||
|
||||
Proofreading by Aminoacyl-tRNA Synthetases Increases the Fidelity of Protein Biosynthesis
|
||||
|
||||
• Threonyl-tRNA synthetase has an editing site that hydrolyzes Serine if this
|
||||
is linked to threonine-tRNA
|
||||
• Because Thr contains an extra methyl group, it is sterically excluded
|
||||
from the editing site
|
||||
• The aminoacylated CCA arm of the tRNA is flexible and can swing out
|
||||
of the activation site and into the editing site to remove Ser
|
||||
• Most aminoacyl-tRNA synthetases contain editing sites and
|
||||
activation sites to ensure very high fidelity.
|
||||
Threonyl-tRNA synthetase Thr-tRNA
|
||||
|
||||
⸻
|
||||
|
||||
Aminoacyl-tRNA synthetases interaction with tRNA
|
||||
|
||||
Threonine-tRNA synthetase
|
||||
tRNA
|
||||
• Threonine-tRNA synthetases bind to both the acceptor stem
|
||||
and the anticodon loop of the tRNA
|
||||
Aminoacyl-tRNA synthetases assign a particular amino acid to a specific tRNA - the true translators of the genetic
|
||||
code
|
||||
• Some synthetases recognize their tRNA partners primarily on
|
||||
the basis of their anticodons
|
||||
• Synthetases may also recognize other
|
||||
aspects of tRNA structure that vary among
|
||||
different tRNAs
|
||||
• many of the recognition sites are loops rich in
|
||||
unusual bases
|
||||
Number of interactions between tRNA
|
||||
and aminoacyl-tRNA synsthetases
|
||||
|
||||
⸻
|
||||
|
||||
The ribosome is the site of protein synthesis
|
||||
|
||||
Ribosomes coordinate the interplay of aminoacyl-tRNAs, mRNA, and proteins
|
||||
aminoacyl-tRNAs
|
||||
|
||||
⸻
|
||||
|
||||
The E. coli ribosome has a sedimentation coefficient of 70S
|
||||
|
||||
composed of:
|
||||
• large (50S) subunit
|
||||
• small (30S) subunit
|
||||
34 proteins (L1–L34)
|
||||
23S rRNA
|
||||
5S rRNA
|
||||
21 proteins (S1–S21)
|
||||
16S rRNA
|
||||
• Two-thirds of the mass of ribosomes is RNA
|
||||
• Ribosomal RNA (rRNA) is the catalyst for protein synthesis
|
||||
|
||||
⸻
|
||||
|
||||
The ribosome has three binding sites for transfer RNAs
|
||||
|
||||
• Three tRNA-binding sites:
|
||||
• A site (aminoacyl)
|
||||
• P site (peptidyl)
|
||||
• E site (exit)
|
||||
• mRNA fragment is bound within the 30S subunit
|
||||
• Each tRNA contacts both 30S and 50S subunits
|
||||
|
||||
⸻
|
||||
|
||||
Overview of mechanism of protein biosynthesis
|
||||
|
||||
Initiation, Elongation, Translocation and Termination
|
||||
Initiation
|
||||
|
||||
⸻
|
||||
|
||||
Initiation of translation
|
||||
|
||||
• Each initiator region usually displays:
|
||||
• start codon: AUG (Met) or sometimes GUG, rarely UUG
|
||||
• purine-rich sequence ~10 nt upstream
|
||||
• Bacterial mRNAs often encode several polypeptides
|
||||
• Shine–Dalgarno sequence = purine-rich region binding rRNA to position initiator codon in P site
|
||||
|
||||
⸻
|
||||
|
||||
Bacterial protein synthesis is initiated by N-formylmethionyl-transfer RNA
|
||||
|
||||
Steps:
|
||||
1. Met linked to tRNAfMet by aminoacyl-tRNA synthetase
|
||||
2. Met amino group is formylated
|
||||
3. fMet-tRNAfMet placed in P site
|
||||
(note: tRNAMet inserts internal Met)
|
||||
|
||||
⸻
|
||||
|
||||
Initiation factors (IF1, IF2, IF3)
|
||||
1. IF1 + IF3 bind 30S to prevent premature binding to 50S
|
||||
2. IF2(GTP) + fMet-tRNAfMet + mRNA bind to 30S → 30S initiation complex
|
||||
3. Structural changes release IF1 + IF3
|
||||
4. IF2 stimulates 50S binding + GTP hydrolysis → 70S initiation complex
|
||||
Streptomycin binds 30S and blocks fMet-tRNAfMet binding (bacteria)
|
||||
|
||||
⸻
|
||||
|
||||
Elongation
|
||||
|
||||
Elongation factors deliver aminoacyl-tRNAs to the ribosome
|
||||
|
||||
⸻
|
||||
|
||||
Elongation factors deliver aminoacyl-tRNAs
|
||||
|
||||
Steps:
|
||||
1. EF-Tu-GTP binds aminoacyl-tRNA → delivers to A site
|
||||
2. Correct codon recognition → GTP hydrolysis → EF-Tu-GDP leaves
|
||||
3. EF-Ts binds EF-Tu-GDP
|
||||
4. EF-Ts releases GDP → GTP binds → EF-Tu-GTP regenerated
|
||||
Tetracycline binds 30S → blocks aminoacyl-tRNA binding
|
||||
|
||||
⸻
|
||||
|
||||
Elongation – peptidyl transferase
|
||||
|
||||
• Amino group of A-site tRNA attacks carbonyl of P-site peptidyl-tRNA
|
||||
• Peptidyl transferase center on 50S catalyzes peptide bond formation
|
||||
• Reaction is thermodynamically spontaneous but accelerated by ribosome positioning/orientation
|
||||
|
||||
⸻
|
||||
|
||||
Translocation
|
||||
|
||||
Translocation repositions tRNAs and mRNA
|
||||
EF-G (translocase) catalyzes 1-codon movement (requires GTP)
|
||||
1. EF-G-GTP binds near A site
|
||||
2. GTP hydrolysis → conformational change → peptidyl-tRNA shifts A→P
|
||||
After peptide bond formation: chain is in P site (50S), anticodon still in A site (30S)
|
||||
|
||||
⸻
|
||||
|
||||
Termination
|
||||
|
||||
Release factors (RFs):
|
||||
• RF1 + RF2 recognize stop codons (UAA, UGA, UAG)
|
||||
• RF3 (GTPase) removes RF1/RF2
|
||||
RF1/RF2 bind stop codon in A site
|
||||
RFs contain conserved GGQ motif with methylated Gln → promotes hydrolysis of ester linkage → releases polypeptide
|
||||
|
||||
⸻
|
||||
|
||||
Bacteria: transcription and translation are coupled
|
||||
|
||||
Minimal time gap between transcription and translation
|
||||
Polysome: multiple ribosomes translating one mRNA simultaneously
|
||||
|
||||
⸻
|
||||
|
||||
Difference between bacterial and eukaryotic protein biosynthesis
|
||||
|
||||
Bacteria 70S: 50S+30S
|
||||
Eukaryotes 80S: 60S+40S
|
||||
Initiation differences:
|
||||
• Initiating amino acid = Met (not fMet)
|
||||
• No Shine–Dalgarno
|
||||
• Start site usually first AUG from 5’ end
|
||||
• Many more eIFs
|
||||
Eukaryotic mRNA circularization: 5’-cap proteins + PABP at 3’-poly(A) interact → circular mRNA
|
||||
|
||||
⸻
|
||||
|
||||
TABLE 30.4 Antibiotic inhibitors of protein biosynthesis
|
||||
|
||||
Streptomycin/aminoglycosides — inhibit initiation + misreading (bacteria)
|
||||
Tetracycline — blocks aminoacyl-tRNA binding (30S)
|
||||
Chloramphenicol — inhibits peptidyl transferase (50S)
|
||||
Cycloheximide — inhibits translocation (eukaryotes)
|
||||
Erythromycin — blocks translocation (50S)
|
||||
Puromycin — premature termination (aa-tRNA analog)
|
||||
|
||||
⸻
|
||||
|
||||
Diphtheria toxin
|
||||
|
||||
Blocks protein biosynthesis in eukaryotes by inhibiting translocation
|
||||
|
||||
⸻
|
||||
|
||||
Concepts
|
||||
|
||||
DNA
|
||||
RNA
|
||||
Protein
|
||||
Translation
|
||||
Transcription
|
||||
Messenger RNA (mRNA)
|
||||
Initiation factors
|
||||
Elongation factors
|
||||
Release factors
|
||||
Aminoacyl tRNA synthetases
|
||||
Transfer RNA (tRNA)
|
||||
Ribosome (ribosomal RNA + proteins)
|
||||
EF1 EF2 EF3
|
||||
EF-Tu EF-Ts EF-G
|
||||
RF1 RF2 RF3
|
||||
• Genetic code
|
||||
• Degeneracy
|
||||
• Codon
|
||||
• Anticodon
|
||||
• Transfer RNA
|
||||
• ’Wobble’ effect
|
||||
• Aminoacyl-tRNA synthetases
|
||||
• Aminoacyl-tRNA
|
||||
• Aminosylation
|
||||
• Aminoacyl-tRNA synthetases activation site
|
||||
• Aminoacyl-tRNA synthetases editing site
|
||||
• Ribosome
|
||||
• Ribosomal RNA
|
||||
• Ribosome E site, P site and A site
|
||||
• Shine-Dalgarno sequence
|
||||
• Initiation, elongation, translocation, termination
|
||||
• Initiation factors
|
||||
• Elongation factors
|
||||
• Termination factors
|
||||
• Polysome
|
||||
• Inhibition of trancription
|
||||
Binary file not shown.
@@ -1,74 +0,0 @@
|
||||
```
|
||||
mRNA kodas i 5'-till-3'-riktning ett kodon i taget
|
||||
Proteinet är syntetiserat från amino- till karboxyl-riktning
|
||||
tRNA är en adaptormolekyl mellan kodonet och aminosyran
|
||||
antikodonet parar med kodonet och är komplementar, innehåller samma information bakvänt
|
||||
För att kunna syntetisera protein måste kodonet läsas av korrekt av antikodonen
|
||||
tRNA är en kedja som innehåller mellan 73 och 93 nukleotider
|
||||
tRNA innehåller 7 till 15 ovanliga baser, metylerade eller demetylerade derivat
|
||||
Sekundärstruktuern av tRNA ser ut som ett klöverlöv
|
||||
Fem grupper av bases i tRNA är inte basparade men deltar i vätebindingar
|
||||
tRNAs sätter fast aminosyror i 3 CCA-terminalaregionen som också heter acceptor stem
|
||||
tRNAs 3' ände har först en adenosine med en hydroxylgrupp som sitter fast med aminosyran
|
||||
tRNAs 3D-struktur ser ut som ett L
|
||||
tRNAs antikodonloop sitter i mitten av sekvensen
|
||||
Genetiska koden är relationen mellan basparen i DNA och sekvensen av aminosyror i proteiner
|
||||
Genetiska koden karakteriseras av: 3 nukleotider (kodon), går i en riktning, överlappar inte, ingen punktuation
|
||||
Den genetiska koden är degenererad: flera kodon kodar för samma aminosyra, vilket minskar effekten av vanliga mutationer
|
||||
Det finns 3 kodon sekvenser som terminerar translationen (UAA, UAG, UGA)
|
||||
61 kodon kodar för 20 aminosyror
|
||||
Vissa tRNA-molekyler kan känna igen fler än ett kodon, det heter Wobble-effekten
|
||||
Den tredje nukleotiden i ett kodon kan ’svaja’ lite, den har större sterisk frihet och behöver inte alltid baspara strikt
|
||||
Svajhypotesen förutser vilket antikodon som kan binda till ett visst kodon
|
||||
Inosin bildas genom deaminering av adenosin och kan bilda vätebindningar med adenin, cytosin och uracil
|
||||
Aminoacyl-tRNA-syntetaser kopplar specifika aminosyror till tRNA
|
||||
När man kopplar en aminosyra till tRNA heter det aminoacylisering
|
||||
Aminosyror kopplar till 2' eller 3'-hydroxylgruppen på det terminala adenosin i tRNA
|
||||
Varje aminosyra har ett specifikt enzym (aminoacyl-tRNA-syntetas), som katalyserar kopplingen av just den aminosyran till rätt tRNA-molekyl.
|
||||
Aminoacyl-tRNA-syntetas har en redigeringsplats som korrekturläser och tar bort felaktigt bundna aminosyror
|
||||
Aminoacyl-tRNA-syntetas har en aktiveringsplats som aktiverar rätt aminosyra genom att binda den till ATP innan den förs över till tRNA.
|
||||
Aktiveringsplatsen försöker välja rätt aminosyra, och redigeringsplatsen fångar upp och tar bort de små fel som ändå passerar vilket ger mycket hög noggrannhet.
|
||||
{{c1::Aminoacyl-tRNA-syntetaser}} är de ”sanna läsarna” av den genetiska koden eftersom de kopplar {{c2::rätt aminosyra rätt tRNA}}, vilket gör att {{c3::kodonet får rätt aminosyra oavsett wobble-variationer}}.
|
||||
Syntetaser kan binda till flera olika igenkänningsställen på tRNA, till exempel acceptorstammen, antikodonloopen eller andra loopstrukturer.
|
||||
De använder olika delar av tRNA för att känna igen rätt tRNA — det är inte alltid samma ställe.
|
||||
Ribosomens stora subenhet heter 50S och består av 34 proteiner
|
||||
Ribosomens lilla subenhet heter 30S och består av 21 proteiner
|
||||
|
||||
Ribosomen katalyseras inte av proteiner utan av {{c1::ribosomalt RNA (rRNA)}}.
|
||||
Peptidbindningen i ribosomen bildas i {{c1::peptidyltransferascentret}}, som består av {{c2::rRNA}}.
|
||||
Ribosomen är ett exempel på en {{c1::ribozym}}, eftersom {{c2::rRNA katalyserar peptidbindningen}}.
|
||||
Ribosomens proteiner fungerar främst som {{c1::strukturellt stöd}}, medan {{c2::rRNA står för katalysen}}.
|
||||
I ribosomen är det {{c1::23S rRNA (bakterier)}} / {{c2::28S rRNA (eukaryoter)}} som utför den katalytiska reaktionen.
|
||||
|
||||
mRNA fragementet binder till den lilla subenheten i ribosomen
|
||||
tRNA rör både stora och lilla subenheten i ribosomen
|
||||
3 tRNA-bindande platser i ribosomen skapar peptidbindingen: Aminoacyl, Peptid och Exit
|
||||
|
||||
Alla mRNA molekyler har en signal som definerar början och slutet på varje polypeptidkedja
|
||||
|
||||
Initieringsregionen i bakteriellt mRNA innehåller vanligtvis ett startkodon och en purinrik sekvens.
|
||||
Shine–Dalgarno-sekvensen är en purinrik region som binder till rRNA och placerar startkodonet i P-siten
|
||||
Bakteriell proteinsyntes initieras av N-formylmethionyl-transfer RNA
|
||||
Bakteriell initierings-tRNA (tRNA^fMet) släpar med N-formylmethionine till ribosomen
|
||||
N-formylmethionyl-tRNA is placed in the P site of the ribosome
|
||||
Initiation factors (IF1, IF2, IF3) assist in the assembly of the protein-synthesizing machinery
|
||||
IF1 and IF3 bind the 30S subunit to prevent premature binding to the 50S subunit
|
||||
IF2(GTP) initiator- fMet-tRNA fMet complex binds with mRNA and the 30S subunit to form the 30S initiation complex
|
||||
70S initiation complex formation is the rate-limiting step in protein biosynthesis
|
||||
Antibiotic Streptomycin Binds to 30S ribosomal subunit and interferes with the binding of fMet-tRNA fMet (Specific to bacteria)
|
||||
Elongation factors deliver aminoacyl-tRNAs to the ribosome
|
||||
Antibiotic Tetracycline Binds to 30S ribosomal subunit and inhibits the binding of aminoacyl-tRNAs (bacteria) Elongation
|
||||
Elongation – peptidyl transferase catalyzes peptide-bond formation
|
||||
peptidyl transferase center = a site on the 50S subunit that catalyzes the thermodynamically spontaneous formation of the peptide bond
|
||||
image av tRNA + beskrivningar för anticodon, CCA, N-term, C-term
|
||||
Translocation repositions tRNAs and mRNA with respect to the ribosome
|
||||
elongation factor G (EF-G, translocase) - catalyzes the movement of mRNA by one codon (requires GTP)
|
||||
Termination is catalyzed by release factors that read stop codons
|
||||
Release factors (RFs) RF1 and RF2 are proteins recognize stop codons (UAA, UGA, or UAG) RF3 is a GTPase that catalyzes the removal of RF1 or RF2 from the ribosome
|
||||
|
||||
```
|
||||
|
||||
Stop 28:55 av https://www.youtube.com/watch?v=gjjp-NUaCXE
|
||||
|
||||
![[Pasted image 20251127131807.png]]
|
||||
|
||||
@@ -1,214 +0,0 @@
|
||||
---
|
||||
tags:
|
||||
- biokemi
|
||||
- translation
|
||||
- undertexter
|
||||
föreläsare: Ana Luis
|
||||
---
|
||||
|
||||
Hello, my name is Anna Lee and in today’s lecture I’ll be talking about protein biosynthesis or translation.
|
||||
|
||||
In previous lectures you have learned that during transcription the RNA polymerase transcribes genes into the messenger RNA or mRNA. In this lecture we’ll cover how the cell translates the mRNA information into proteins.
|
||||
|
||||
The main learning goals of this lecture are:
|
||||
- explaining how nucleic acid information is translated into amino acid sequence
|
||||
- defining the role of aminoacyl tRNA synthetases in this process
|
||||
- understanding the features of the genetic code
|
||||
- understanding the structure and function of tRNA
|
||||
- understanding the structure and function of ribosomes
|
||||
- understanding the steps of protein synthesis
|
||||
- understanding how antibiotics and toxins block protein biosynthesis
|
||||
|
||||
Before we start with the details of translation, consider the question: why is it important to understand the mechanisms of translation?
|
||||
|
||||
Reasons:
|
||||
- inhibition of translation of specific mRNAs can lead to diseases
|
||||
- example: fragile X mental retardation syndrome
|
||||
- translation is targeted by bacterial toxins
|
||||
- example: diphtheria toxin
|
||||
- translation is a major target of antibiotics
|
||||
- examples: streptomycin, tetracycline
|
||||
- new antibiotics are being developed that target bacterial ribosomes
|
||||
|
||||
---
|
||||
|
||||
Protein biosynthesis is called translation because:
|
||||
- the four-letter nucleic acid alphabet is translated into the 20-letter amino acid alphabet
|
||||
- this conversion makes translation more complex than replication or transcription
|
||||
|
||||
General principles:
|
||||
- mRNA is decoded 5′→3′
|
||||
- protein is synthesized from amino (N) to carboxyl (C) terminus
|
||||
- amino acids are added sequentially to the C-terminus
|
||||
|
||||
Translation requires:
|
||||
- a codon (three RNA bases)
|
||||
- a tRNA acting as an adaptor
|
||||
- correct codon–anticodon recognition
|
||||
|
||||
---
|
||||
|
||||
tRNA molecules:
|
||||
|
||||
Common features:
|
||||
- contain 7–15 modified bases
|
||||
- secondary structure resembles a cloverleaf
|
||||
- five major regions:
|
||||
- 3′ CCA acceptor stem
|
||||
- TΨC loop
|
||||
- extra arm
|
||||
- anticodon loop
|
||||
- DHU loop
|
||||
- 3D structure is L-shaped
|
||||
- anticodon is positioned ~90° from the 3′ CCA end
|
||||
- the CCA region can undergo conformational changes
|
||||
|
||||
---
|
||||
|
||||
The genetic code:
|
||||
|
||||
Key characteristics:
|
||||
- codons are groups of three nucleotides
|
||||
- the code is read 5′→3′
|
||||
- non-overlapping
|
||||
- no punctuation
|
||||
- degenerate (most amino acids have multiple codons)
|
||||
- three stop codons
|
||||
|
||||
The wobble effect:
|
||||
- some tRNAs recognize more than one codon
|
||||
- the third codon base is less strictly recognized
|
||||
- wobble depends on the first base of the anticodon
|
||||
|
||||
---
|
||||
|
||||
Aminoacyl tRNA synthetases:
|
||||
|
||||
Functions:
|
||||
- attach amino acids to the correct tRNAs
|
||||
- establish the genetic code
|
||||
- activate amino acids with ATP
|
||||
- form an ester bond with the 3′ end of tRNA
|
||||
|
||||
Fidelity mechanisms:
|
||||
- activation site
|
||||
- editing (proofreading) site
|
||||
|
||||
Example: threonine vs valine vs serine
|
||||
- the enzyme’s zinc-dependent binding site excludes valine
|
||||
- serine can bind but is removed in the editing site
|
||||
|
||||
tRNA recognition:
|
||||
- often involves acceptor stem and anticodon loop
|
||||
- sometimes additional structural features
|
||||
|
||||
---
|
||||
|
||||
Ribosomes:
|
||||
|
||||
In bacteria:
|
||||
- 70S ribosome
|
||||
- 50S large subunit (23S rRNA, 5S rRNA, proteins L1–L34)
|
||||
- 30S small subunit (16S rRNA, proteins S1–S21)
|
||||
|
||||
Functional sites:
|
||||
- A site (aminoacyl)
|
||||
- P site (peptidyl)
|
||||
- E site (exit)
|
||||
|
||||
---
|
||||
|
||||
Translation stages:
|
||||
- initiation
|
||||
- elongation
|
||||
- translocation
|
||||
- termination
|
||||
|
||||
---
|
||||
|
||||
Initiation in bacteria:
|
||||
|
||||
Requirements:
|
||||
- start codon (usually AUG)
|
||||
- Shine–Dalgarno sequence upstream
|
||||
- base-pairs with 16S rRNA
|
||||
- positions start codon in P site
|
||||
|
||||
Initiator:
|
||||
- formyl-methionine (fMet)
|
||||
- delivered by fMet-tRNAᶠᴹᵉᵗ
|
||||
|
||||
Initiation factors:
|
||||
- IF1 and IF3 bind the 30S subunit to prevent premature 50S binding
|
||||
- IF2-GTP recruits fMet-tRNAᶠᴹᵉᵗ
|
||||
- GTP hydrolysis allows 50S binding and forms 70S initiation complex
|
||||
|
||||
This step is a major regulatory point and is targeted by antibiotics such as streptomycin.
|
||||
|
||||
---
|
||||
|
||||
Elongation:
|
||||
|
||||
- EF-Tu-GTP delivers aminoacyl tRNA to the A site
|
||||
- correct codon recognition → GTP hydrolysis → EF-Tu-GDP release
|
||||
- EF-Ts regenerates EF-Tu-GTP
|
||||
|
||||
Peptide bond formation:
|
||||
- catalyzed by the 23S rRNA peptidyl transferase center
|
||||
- the amino group in the A site attacks the ester bond in the P site
|
||||
|
||||
Translocation:
|
||||
- driven by EF-G-GTP
|
||||
- tRNAs shift A→P and P→E
|
||||
- empty tRNA exits
|
||||
|
||||
---
|
||||
|
||||
Termination:
|
||||
|
||||
- no tRNAs recognize stop codons
|
||||
- RF1 or RF2 bind stop codons in the A site
|
||||
- RF3-GTP triggers release
|
||||
- the polypeptide is freed from the P-site tRNA
|
||||
|
||||
---
|
||||
|
||||
Coupling in bacteria:
|
||||
|
||||
- transcription and translation occur simultaneously
|
||||
- multiple ribosomes can translate the same mRNA (polysomes)
|
||||
|
||||
---
|
||||
|
||||
Eukaryotic translation:
|
||||
|
||||
Differences:
|
||||
- ribosomes are larger (80S = 60S + 40S)
|
||||
- initiating amino acid is methionine (not fMet)
|
||||
- no Shine–Dalgarno sequence
|
||||
- the start codon is identified as the first AUG near the 5′ end
|
||||
- eukaryotic mRNA is processed before translation
|
||||
- mRNA circularizes via 5′ cap–poly(A) interactions
|
||||
|
||||
---
|
||||
|
||||
Drugs and toxins affecting translation:
|
||||
|
||||
Examples:
|
||||
- streptomycin: inhibits initiation in bacteria
|
||||
- tetracycline: blocks aminoacyl tRNA binding
|
||||
- cycloheximide: inhibits eukaryotic elongation
|
||||
- puromycin: chain terminator in both systems
|
||||
- diphtheria toxin: blocks eukaryotic translocation
|
||||
|
||||
---
|
||||
|
||||
This lecture covered:
|
||||
- molecules involved in translation
|
||||
- features of the genetic code
|
||||
- ribosome structure
|
||||
- steps of translation
|
||||
- differences between bacterial and eukaryotic translation
|
||||
- drugs and toxins that block protein biosynthesis
|
||||
|
||||
If you have any questions, feel free to contact me via email or in Canvas.
|
||||
Reference in New Issue
Block a user