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526-301 Biotechnology
TRANSLATION
Dr Mike Dyall-Smith, 2007
526-301 Biotechnology
Protein synthesis in bacteria
Dr Mike Dyall-Smith, lab 3.07, [email protected]
Aims:
• Understand the process of translation in Bacteria
• Know the the major components involved
• Know the typical genetic signals needed to allow
translation of a mRNA
• Familiar with the process of protein folding
References:, Schaecter et al., Microbe, pp149-58 or Any
recent molecular biology textbook. A recent review is:
http://mmbr.asm.org/cgi/content/full/69/1/101#Initiator_tRNA
Dr Mike Dyall-Smith, 2007
526-301 Biotechnology
Lecture outline:
1. Overview of translation in bacteria (comparison with
eucarya) components, genetic code, process.
2. Features of mRNA and tRNA
4. Ribosome structure and function (compared to
eucarya)
5. Initiation and termination factors and the process
of translation
6. Briefly touch on protein folding. *export and
processing covered in future lecture.
Dr Mike Dyall-Smith, 2007
526-301 Biotechnology
Central dogma
DNA
Transcription
RNA
Translation
PROTEIN
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Dr Mike Dyall-Smith, 2007
Translation
The decoding (translation) of the 3-letter
code of nucleotide bases in the mRNA, to
direct the synthesis of proteins.
A much more complex operation than
transcription.
526-301 Biotechnology
Overall process of translation in bacteria
Initiation factors
Requires
aa-tRNAs,
ATP, GTP
Speed:
~ 15 aa/sec
mRNA
Initiation Elongation Termination
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Dr Mike Dyall-Smith, 2007
526-301 Biotechnology
Protein synthesis in bacteria
How do ribosomes bind to mRNA?
• Small subunit binds initiation factors
• This complex binds to mRNA, initiator
tRNA can then bind.
• The large subunit joins the complex
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Amino acid
added at 3’ end
526-301 Biotechnology
Transfer RNA
Acceptor Arm
D Arm
T Arm
Extra Arm
Anticodon
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526-301 Biotechnology
Transfer RNA (tRNA) structure
Linkage to aa
Arm
tRNA
Anticodon
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526-301 Biotechnology
tRNA is actually ‘L’ shaped
Anticodon arm
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526-301 Biotechnology
UNIVERSAL GENETIC CODE
Picture from Schaecter et al.
For use by students enrolled in 526-301
Dr Mike Dyall-Smith, 2007
526-301 Biotechnology
Ribosomes are large enough to bind 2
tRNAs and cover 40 nt of mRNA
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For use by students enrolled in 526-301
Dr Mike Dyall-Smith, 2007
526-301 Biotechnology
Pictures mainly from Genes V (Lewin)
For use by students enrolled in 526-301
Dr Mike Dyall-Smith, 2007
526-301 Biotechnology
Pictures mainly from Genes V (Lewin)
For use by students enrolled in 526-301
Dr Mike Dyall-Smith, 2007
526-301 Biotechnology
70 S Ribosomes
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Dr Mike Dyall-Smith, 2007
Ribosomes contain many
proteins and 3 rRNAs
2/3 of the mass of bacterial ribosomes is rRNA
Initiation of translation:
Initiation of translation:
IF-2 puts the fMet-tRNAf initiator
in the P site. IF-2, bound to GTP,
associates with the P site of the
30S subunit.
fMet-tRNAf. then binds to the IF2 on the 30S subunit.
IF-2 then transfers the tRNA into
the partial P site
50S subunit binds and IF1-3 are
released.
526-301 Biotechnology
Where do ribosomes bind on mRNA ?
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526-301 Biotechnology
Ribosome binding
sites bind to the 3’
end of 16S rRNA
Shine-Dalgarno
sequence
QuickTime™ and a
TIF F (Uncompressed) decompressor
are needed to see this picture.
Also called ribosome
binding site or RBS
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Initiator tRNA in bacteria is tRNAf MET
N-formyl-methionyl tRNA. It is only used for
initiating translation. All proteins start with this
amino acid. Internal methionines use a different
tRNA, tRNAmMET
526-301 Biotechnology
tRNA and mRNA move in the same direction
through the ribosome
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A site : site where an aminoacyl-tRNA enters to base pair
with a codon.
P site : site occupied by a peptidyl-tRNA
Deacylated tRNA: has no amino acid or polypeptide chain
attached
Translocation: the movement of the ribosome, one codon
at a time, along mRNA after the addition of an amino acid to
the polypeptide chain.
Elongation: the stage in a macromolecular synthesis
reaction (replication, transcription, or translation) when the
nucleotide or polypeptide chain is extended by the addition
of individual subunits.
526-301 Biotechnology
Two sites for binding
tRNAs on the ribosome
P A
Ribosome movement
P and A sites occupied
Peptide bond formed
Translocation
Dr Mike Dyall-Smith, 2007
526-301 Biotechnology
Typical polycistronic bacterial mRNA
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526-301 Biotechnology
Reinitiation on polycistronic mRNA
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Termination at stop codons:
UAA, UGA, UAG
Termination codons are recognised by
‘release factor’ proteins (RF1/RF2, RF3).
RF1 or RF2 bind to stop codons in the A
site of ribosomes, and activate the
ribosome to hydrolyse the adjacent
peptidyl tRNA (in the P site), to release
the protein.
RF3 releases the RF1 or RF2.
Finally, RRF (ribosome recycling factor)
dissociates the remaining mRNA, tRNA
and ribosome subunits.
526-301 Biotechnology
mRNA degradation in bacteria is rapid
Half-life of 1-3 min
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526-301 Biotechnology
mRNA degradation in bacteria is rapid
Q: What happens if the ribosome reaches
the end of a broken mRNA without seeing
a stop codon ? A: it gets stalled !!
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Dr Mike Dyall-Smith, 2007
526-301 Biotechnology
tmRNA - how to overcome stalled ribosomes
tmRNA is part tRNA part
mRNA. It completes
translation, putting a small
peptide sequence at the end
of a protein. This is a tag for
protein degradation.
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
http://www.indiana.edu/~tmrna/
Pictures mainly from Genes V (Lewin)
For use by students enrolled in 526-301
Dr Mike Dyall-Smith, 2007
526-301 Biotechnology
Overall process of translation in Bacteria
Initiation factors
Requires
aa-tRNAs,
ATP, GTP
Speed:
~ 15 aa/sec
mRNA
Initiation Elongation Termination
Pictures mainly from Genes V (Lewin)
For use by students enrolled in 526-301
Dr Mike Dyall-Smith, 2007
Protein folding
• Most proteins will not fold correctly
without assistance
• Cell has several chaperones that
perform that function
• Otherwise, misfolded proteins are
degraded by proteases
Protein folding
• Trigger factor: peptidyl proline isomerase
• DnaK: removes small, misfolded
hydrophobic regions
• GroEL and GroES: major
folding/refolding, protected from
proteases
Protein folding
526-301 Biotechnology
Lecture outline:
1. Overview of translation in bacteria (and comparison
with eucarya) components, genetic code, process.
2. Features of mRNA and tRNA
3. Ribosome structure and function (compared to
eucarya)
4. Initiation and termination factors and the process
of translation
5. Outline of protein folding
Pictures mainly from Genes V (Lewin)
For use by students enrolled in 526-301
Dr Mike Dyall-Smith, 2007