Part B

Molecular Biology [012] Proteins

DNA

RNA
Finn Werner
Director, Institute of
Structural and Molecular Biology
UCL, London, UK
protein
Three languages or codes in Biology ~1960 AD

DNA
Jim Watson

Francis Crick

RNA
The central dogma of
molecular biology – ‘DNA makes
RNA makes protein’
Protein
 Mechanisms of translation

Initiation
- Or how to recognise the start of an open reading
frame (ORF)
 Bacteria

5’ AUG GGG AAA GCA UUU CUG AUG CGC ACU UCC CCC UGA 3’

leader AUG UGA trailer

Direct baseparing
between ribosomal
RNA and mRNA
RBS

mRNA 5’ GGAGGA(T/A)9 AUG 3’

CCUCCU
16S rRNA
3’ 5’

RBS = ribosome binding site also ‘Shine-Dalgarno’ sequence

 Eukaryotes
5’ GGG AAA GCA CCACC AUG CGC ACU AUG UCC CCC UGA 3’

5’ UTR AUG UGA 3’ UTR

UTR = untranslated region

Not facilitated by
Kozak sequence (on mRNA) direct baseparing
CCACCAUGG between ribosomal
RNA and mRNA

5’ AUG UGA AAA 3’

cap
mRNA
 Similar to nucleic acid polymerases
Ribosomes require accessory factors to function
The Other Players
(1) Initiation factors: " IF1
" " " " IF2 (GTPase)
" " " " IF3
(2) Elongation factors:" EF-Tu (GTPase)
" " " " EF-G (GTPase)
(3) Termination factors:" RF-1
(Release factors)" RF-2
" " " " RF-3 (GTPase)
" " " " RRF (Ribosome recycling factor)

Energy released by the hydrolysis of GTP facilitates

conformational (structural) changes – the fuel of the engine
Initiation

1. mRNA CCUCC
GGAGG AUG

GTP
IF3 IF1 IF2
2. GGAGG AUG

IF=initiation factor
 fmet GTP
3.
IF3 IF1 IF2
GGAGG AUG

30S initiation complex (mRNA+small subunit)

GDP +Pi
4. P A
fmet Initiation
E
Factors
GGAGG AUG released

70S initiation complex (mRNA+small+large subunit)

P = peptidyl-tRNA binding site

A = aminoacyl-tRNA binding site
E = tRNA exit site
Mechanisms of translation

Elongation
- How to move along the mRNA
 Translation elongation I
Overview of decoding and elongation I

• tRNA-aa binds reversible as a complex with EF-Tu*GTP

• Anticodon can interact with codon in the A-site, CCA-end
with bound aa is bound to EF-Tu (A/T-state)
• GDP bound EF-Tu dissociates rapidly from ribosome
Overview of decoding and elongation
Translation elongation II
II
• Accommodated tRNA*aa rapidly undergoes peptidyl-transferase
reaction - aa added to the polypeptide chain
• Ribosome must shift the mRNA to the 3’
• EF-G*GTP binds to the ribosome
• GTPase activity generates EF-G*GDP, translocates the peptidyl
tRNA from the A to the P site and the deacylated tRNA shifts to E
• EF-G*GDP then dissociates from the ribosome
Mechanisms of translation
 A reoccurring theme

Baseparing and nucleotides

• Replication template
• Replication energy substrates
• Transcription template
• Transcription energy substrates
• Translation decoding mRNA-tRNA
• Translation ribosome-mRNA binding
• Translation factor energy substrates

Nucleic acids – the deep fabric of

biological information
Application of the knowledge about
ribosome structure and function
The Good - Antibiotics fight bacterial infection

• Antibiotics are made by funghi in nature

• Means to eliminate ‘the competition’ for resources
• Target vital processes in the cell
(cell division, transcription and translation)

tetracycline - inhibits aminoacyl-tRNA binding

erythromycin - binds peptidyl transferase site and blocks

polypeptide exit from ribosome

chloramphenicol - blocks correct positioning of the

aminoacyl-tRNA inhibits peptidyl transferase reaction
 The Bad – toxins as bioweapons and disease
Ricin - targets eukaryotic and prokaryotic cells -
prevents activation of translation factor GTPases

lys GTP
P A
fmet X EF-Tu X
AUG AAA GGG
GDP +Pi
• The umbrella murder of a Bulgarian spy in ’78
• Breaking Bad’s Heisenberg poison
• Recent terrorist caught by FBI for attempting to buy ricin on the internet

Diptheria toxin - acts on a eukaryotic elongation factor

The toxin couples ADP-ribose to the factor and inactivates it

EF
[012] A large array of translation factors facilitate translation initiation
and elongation, many of which utilise GTPase activity to induce
conformational changes such as the translocation movement of the
ribosome along the mRNA. Inhibitors of ribosomes can be useful (eg.
Antimicrobial antibiotics) or harmful (eg. Pathogen toxins). Genetic
mutations can originate from frameshift, missense or nonsense
mutations.