Translation & Proteins II Flashcards
Translation
Consists of various steps which can be divided into 3 basic processes: Initiation, elongation and termination.
But remember it is a continuous dynamic process - not in separate steps
Translation
Components:
- Large and small ribosomal subunits
- mRNA
- Charged tRNAs
- GTP
- Mg++
- Initiation factors (IF1, 2, 3)
- Elongation factors (EF-Tu, EF-G)
Initiation ( in prokaryotes)
- When ribosomes aren’t actually involved in translation - they
dissociate into their large and small subunits
- Initiation starts with the small 30s subunit binding to 3 initiation factors (IF) - which enhance the binding of other components
- This complex then binds to the mRNA at a specific sequence
= Shine-Dalgarno sequence
(AGGAGG - contains only purines and is before AUG (start
codon) )
- In prok. this complex is actually the binding site for ribosomes
- 16s rRNA whose function is to recognize the mRNA
> IF2 enhances the binding of tRNA which carries formyl methionine to small ribosomal subunit - in response to the start codon complex and thus sets reading frame for the rest of the translation process
> IF3 plays a role in dissociating the 2 ribosomal sub-units
following termination is then released
- with the release of IF 3 - the complete Initiation complex
binds with large ribosomal subunit - Process uses GTP which is hydrolyzed – provides energy.
- remaining 2 IFs (1 and 2) are released
- As the two ribosomal subunits bind, two binding sites for
charged tRNA molecules are formed
(P=peptidyl sites and A=aminoacyl sites)
Elongation
- at end of Initiation - charged tRNA (baring Fmet) bound in P site
- 2nd tRNA bound above A site; and determines which amino acid will be incorporated in the coming elongation phase
> Binding of this next aminoacyl tRNA (charged tRNA) to this complex is facilitated by IF - EF-Tu - Covalent bond between tRNA and its P-site and its amino acid is broken and uncharged tRNA is released from P site – 1st cycle completed
- a peptide bond between the two amino acids is formed by the catalytic activity of 23S rRNA of the large subunit
= peptidyl transferase activity - ribozyme
(because involves RNA and not protein) - The dipeptide that is formed remains attached to the tRNA,
which is currently sitting in the A-site - once released from its amino acid - Uncharged tRNA from
the p site moves into E (exit) site.
= Ribosomal complex translocates three bases to the right
(direction of P) - but tRNA moves to the left - From A site, to P, to E
> Third codon now positioned above A site and is ready to
accept the third charged tRNA - This Process requires participation of several EFs
- Energy through hydrolysis of GTP is also used to lock all the
proper structures into place - Process repeats itself to form peptide chain
30S subunit - decode codons by holding them over the A site
50S subunit – catalyze peptide bonding - via 23s rRNA unit
-The peptide then exits the ribosome through a tunnel in the large subunit
> Polypeptide (~30 aa) exits base of 50S subunit - visible
as it leaves
Rate = 15 aa/sec; error rate 10-4 - in E.coli at 37 degrees C - This process continues until a termination codon reaches the
A site
Termination
Termination codon (UAG, UAA, UGA)
- Don’t specify an amino acid and have no corresponding tRNAs
- When they appear in A site = signals termination
- often an mRNA has several consecutive termination codons
- The peptide will remain attached to the tRNA in the p site, but
the A site will be empty
- This signals the action of the GTP-dependent release factors
which cleave polypeptide chain from terminal tRNA and
ribosome
- tRNA released from ribosome
- Ribosomal complex dissociates into subunits
- Polypeptide folds into tertiary protein structure
Polysomes
- Once elongation has proceeded beyond the initial portion of
the mRNA, the Shine-Dalgarno sequence is then free to
associate with another small sub-unit to form a second initiation
complex - This process can be repeated many times along the length of
the mRNA = Polysome = mRNA with many monosomes - Polysomes allow for the most efficient use of the components available for the translation per unit time
Where would the Shine-Dalgarno sequence be
On the side with the shorter amino acid chains
Translation in Eukaryotes
- Ribosomes larger and more complex
> contain more rRNA and more proteins - rRNA more complex (share core sequences with prok. but have
expansion sequences which give additional functionality) - Transcription and translation spatially and temporally separated
(unlike prok.) - Transcription takes place in nucleus (not cytoplasm) and the
mRNA must first be processed and then moved out into the
cytoplasm for translation
> this offers enhanced options for gene regulation and
expression
Differences in mRNA: - mRNA has a 5’ 7-mG cap – essential for effective initiation
> 5’ cap = Ribosome binding site - Once bound, the ribosome then recognises the Kozak
consensus (A or GNNAUGG) - similar to shine-Dalgarno
sequence, except for the fact that the S-D seq. in prok acts as
the ribosome binding site - The Kozak consensus represents purines ( A or G). and sits 3
base pairs up from start codon - mRNA has a poly(A) tail – closed-loop translation
> essential for mRNA stability and non-poly (A) mRNAs are
rapidly degraded by nucleases in the cytoplasm - The poly (A) tail plays an NB role in translation initiation
- These proteins are also able to bind to an euk. IF - eIF4G -
which in turn binds to cap-binding protein eIF4E which is
bound to the 5’ cap - This resulting complex is needed for initiation in many euk.
mRNAs - Such closed-loop translation may be advantageous as it
prevents the waste of resources from translating any partially
degraded mRNA which lack either a cap or a poly A - Such a system may also allow ribosome recycling
Translation in Eukaryotes - continued
- Fmet is needed for initiation of translation in prok.
- In euk. the AUG start codon incorporates normal methionine
HOWEVER: at initiation, this methionine is attached to unique tRNA = tRNAi - tRNAi carrying methionine, reacts with eukaryotic IF2 (eIF2) -
which is already bound to GTP to form a tertiary complex
= 1st step in the initiation of protein synthesis - Euk. mRNA live longer than prok. mRNA - hours rather than
minutes = they are available for much longer periods for
protein synthesis - More complex protein factors are needed for translation
- 9 IFs
- Ribosomes in prok. are found free in the cytoplasm - whereas ribosomes in euk. are associated with the membranes of endoplasmic reticulum (ER)
= Modification and transport of newly made proteins
Translation process in Eukaryotes
- Met-tRNAi binds to GTP and eIF2
- Complex binds to small subunit-this needs elF4 to bind to 5’-
cap of mRNA - Complex “scans” RNA sequence to find initiation codon
(Kozak sequence – A/GCCAUGG) - Anticodon base pairs with initiation codon
- Large subunit binds
- Initiation factors released; GTP hydrolized for energy
- Met-tRNAi in P site, 2nd codon in A
- Elongation and termination are very similar
(names and number of protein factors differ).
Proteins - early studies
Garrod & Bateson (1903):
Pathogens are not the cause of all diseases
Some diseases can have a genetic origin
Exemplified by metabolic defects that manifest in the phenotype.
= This implies that heredity info controls chemical reactions in
body
- Proposed relationship between genes (unit factors) and
enzymes (ferments)
Phenylketonuria and alkaptonuria
Experiments by Beadle & Ephrussi (1933) and Beadle & Tatum (1940)
- Provided First evidence that genes are responsible for the production of enzymes.
- Used Drosophila eye pigment and Neurospora mutants
respectively - Change in eye colour could be linked to biochemical alterations involving the loss of enzyme function
set the foundations for the one gene, one enzyme hypothesis.
Neurospora crassa
listen slide 18 on translprot 2
Proteins - early studies
- Methods used for Neurospora study later used to investigate
metabolic paths in various other organisms as well. - Refinement of one gene, one enzyme hypothesis leads to one
gene, one protein …and later to one gene, one polypeptide
hypothesis. - wasn’t clear how a mutation in a single enzyme could cause
variation in so many different types of phenotypic traits - Later it became clear that not all proteins are enzymes
One gene, one polypeptide hypothesis resulted from a study on sickle cell anemia, which is caused by a hemoglobin mutation.
Sickle cell anaemia
- Red blood cells become elongated and curved at low O2
levels because of the polymerization of hemoglobin levels
Slides 20 and 21
