Download Streptococcus pyogenes - Mike Dyall

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.
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
Pictures mainly from Genes V (Lewin)
For use by students enrolled in 526-301
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
Pictures mainly from Genes V (Lewin)
For use by students enrolled in 526-301
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
Pictures mainly from Genes V (Lewin)
For use by students enrolled in 526-301
Dr Mike Dyall-Smith, 2007
526-301 Biotechnology
Transfer RNA
Acceptor Arm
D Arm
T Arm
Extra Arm
Anticodon
Pictures mainly from Genes V (Lewin)
For use by students enrolled in 526-301
Dr Mike Dyall-Smith, 2007
526-301 Biotechnology
Transfer RNA (tRNA) structure
Linkage to aa
Arm
tRNA
Anticodon
Pictures mainly from Genes V (Lewin)
For use by students enrolled in 526-301
Dr Mike Dyall-Smith, 2007
526-301 Biotechnology
tRNA is actually ‘L’ shaped
Anticodon arm
Pictures mainly from Genes V (Lewin)
For use by students enrolled in 526-301
Dr Mike Dyall-Smith, 2007
526-301 Biotechnology
UNIVERSAL GENETIC CODE
Pictures mainly from Genes V (Lewin)
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
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
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
Pictures mainly from Genes V (Lewin)
For use by students enrolled in 526-301
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 ?
Pictures mainly from Genes V (Lewin)
For use by students enrolled in 526-301
Dr Mike Dyall-Smith, 2007
526-301 Biotechnology
Ribosome binding
sites bind to the 3’
end of 16S rRNA
Shine-Dalgarno
sequence
RBS or SDS
Pictures mainly from Genes V (Lewin)
For use by students enrolled in 526-301
Dr Mike Dyall-Smith, 2007
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 another
tRNA, tRNAmMET
526-301 Biotechnology
tRNA and mRNA move in the same direction
through the ribosome
Pictures mainly from Genes V (Lewin)
For use by students enrolled in 526-301
Dr Mike Dyall-Smith, 2007
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
Pictures mainly from Genes V (Lewin)
For use by students enrolled in 526-301
Dr Mike Dyall-Smith, 2007
526-301 Biotechnology
Reinitiation on polycistronic mRNA
Pictures mainly from Genes V (Lewin)
For use by students enrolled in 526-301
Dr Mike Dyall-Smith, 2007
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
Pictures mainly from Genes V (Lewin)
For use by students enrolled in 526-301
Dr Mike Dyall-Smith, 2007
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 !!
Pictures mainly from Genes V (Lewin)
For use by students enrolled in 526-301
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
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
526-301 Biotechnology
Signal sequence of HGH
This is removed (by the pituitary cells) during
secretion, to form the mature hormone.
Dr Mike Dyall-Smith, 2007
526-301 Biotechnology
Review of Paper:
Dr Mike Dyall-Smith, 2007
526-301 Biotechnology
ABSTRACT
Goeddel et al., Nature 281:544 (1979)
Dr Mike Dyall-Smith, 2007
526-301 Biotechnology
Structure of pig growth hormone.
alpha helix
N
C
S-S bond
191 aa
S.Abdel-Meguid, Monsanto Co.
Dr Mike Dyall-Smith, 2007
526-301 Biotechnology
Signal sequence of HGH
This is removed (by the pituitary cells) during
secretion, to form the mature hormone.
Dr Mike Dyall-Smith, 2007
526-301 Biotechnology
Problems in expressing a eukaryotic
protein like HGH in a prokaryote like E.coli
• HGH is normally produced as a pre-protein,
with a signal sequence (for secretion) that is
cut off on export from the cell.
• Need to get rid of introns
• Need to supply E.coli sequences for
transcription and translation.
Goeddel et al., Nature 281:544 (1979)
Dr Mike Dyall-Smith, 2007
526-301 Biotechnology
Strategy for cloning and expressing HGH
• Construct a gene that will express the
mature form of HGH (ie. no signal
sequence)
• Combine cDNA and synthetic DNA to
construct the ORF so that the ATG is
perfectly positioned with a lac promoter.
Goeddel et al., Nature 281:544 (1979)
Dr Mike Dyall-Smith, 2007
526-301 Biotechnology
Construction of pHGH107
2x lacUV5 promoters
Goeddel et al., Nature 281:544 (1979)
HGH and
TetR genes
transcribed
together
Dr Mike Dyall-Smith, 2007
526-301 Biotechnology
Adjusting the distance between the
Shine-Dalgarno sequence and the
start codon.
In the cloning steps, the spacing was 11bp. This
was reduced to 7bp, as in the natural lacZ gene.
Goeddel et al., Nature 281:544 (1979)
Dr Mike Dyall-Smith, 2007
526-301 Biotechnology
Expression of HGH in E.coli
Note the mid-log and stationary phase
levels differ. Levels are lower in stationary
phase cells.
Goeddel et al., Nature 281:544 (1979)
Dr Mike Dyall-Smith, 2007
526-301 Biotechnology
Expression of HGH in E.coli
pHGH107-1 has the 7bp spacing between SD and ATG (as in lacZ)
It is worse than the 11bp version!
Goeddel et al., Nature 281:544 (1979)
Dr Mike Dyall-Smith, 2007
526-301 Biotechnology
Expression of HGH in E.coli
LacI overproducer strain (D1210) shows good
control of gene expression (+/- IPTG inducer).
Goeddel et al., Nature 281:544 (1979)
Dr Mike Dyall-Smith, 2007
526-301 Biotechnology
HGH production in E.coli
- analysed by SDS-PAGE
purification from E.coli
immuno precipitation
pure
HGH
Multiple
bands ??
Goeddel et al., Nature 281:544 (1979)
Dr Mike Dyall-Smith, 2007
526-301 Biotechnology
Summary: What they achieved.
“This is the first time that a human polypeptide has
been directly expressed in E.coli in a non-precursor
form.”
•Made a gene hybrid (cDNA and synthetic DNA) that
coded for the processed form of HGH.
•Able to express HGH protein in E.coli at high levels
(could purify it easily)
•Expressed HGH was able to react with anti-HGH
antibodies
Goeddel et al., Nature 281:544 (1979)
Dr Mike Dyall-Smith, 2007
526-301 Biotechnology
Summary: What they didn’t achieve.
• Did it contain f-Met or not?
• Was it folded correctly? Did it have the
required S-S bridges?
• Was it active as a growth hormone?
• Why was there so much proteolytic
degradation?
Goeddel et al., Nature 281:544 (1979)
Dr Mike Dyall-Smith, 2007
526-301 Biotechnology
Fig. 1. Construction of
plasmid pHGH31,
containing the coding
sequence (cDNA) for
amino acids 24-191 of
Human Growth Hormone.
Goeddel et al., Nature 281:544 (1979)
Dr Mike Dyall-Smith, 2007
526-301 Biotechnology
Plasmid
cloning
vector
cDNA
copy of
HGH
mRNA
Cut in the
middle of the
AmpR gene
Tailed
cut
ends
with
poly C
Tailed 3’
ends with
poly G
Goeddel et al., Nature 281:544 (1979)
Dr Mike Dyall-Smith, 2007
526-301 Biotechnology
Fig 1.
E.coli
Cloned HGH
gene using
polyC-polyG
‘sticky ends’
HaeIII and
PstI sites
regenerated
Goeddel et al., Nature 281:544 (1979)
Dr Mike Dyall-Smith, 2007
526-301 Biotechnology
Sequence of the Human GH cDNA
Goeddel et al., Nature 281:544 (1979)
Dr Mike Dyall-Smith, 2007
526-301 Biotechnology
Construction of the
sequence coding
for aa 1-24 of
mature HGH
Dr Mike Dyall-Smith, 2007
526-301 Biotechnology
Construction of the coding sequence for the
first 24aa of mature HGH
Small synthetic DNA sequences were
assembled and ligated together. The terminal
oligos contained restriction site ‘sticky ends’.
Goeddel et al., Nature 281:544 (1979)
Dr Mike Dyall-Smith, 2007
526-301 Biotechnology
Combining the DNA segments
coding for aa 1-24 and aa 24-191
Goeddel et al., Nature 281:544 (1979)
Dr Mike Dyall-Smith, 2007