MOLECULAR BIOLOGY – Protein synthesis

MOLECULAR BIOLOGY – Protein synthesis
The nucleotide sequence of mRNA contains three letter codons that specify all of the 20 amino acids found in proteins plus a signal to terminate protein synthesis
The order that the codons appear in the mRNA (5’ - 3’) directly dictates the order of the amino acids in the polypeptide chain of the protein (N - C termini)
Figure 6-50 Molecular Biology of the Cell (© Garland Science 2008)

MOLECULAR BIOLOGY – Protein synthesis
+1 frameshift
+2 frameshift
The genetic information encoded in each reading frame is different
Figure 6-51 Molecular Biology of the Cell (© Garland Science 2008)

MOLECULAR BIOLOGY – Protein synthesis
How is the mRNA genetic code read during protein synthesis?
Transfer RNAs (tRNA) act as adapters between the mRNA and protein synthesising machinery (‘ribosomes’)
As each specific tRNA ( i.e.
defined by its anticodon) is bound to a specific amino acid at its 3’ end, according to the genetic code in the mRNA, is recruited to the ribosome tRNA triplet nucleotide sequences that are complementary to mRNA codons, called ‘anticodons’, form specific base-pairs with the mRNA codons
Figure 6-52 Molecular Biology of the Cell (© Garland Science 2008)

MOLECULAR BIOLOGY – Protein synthesis
The minimum set of required tRNAs is 31 but there are 61 possible amino acid coding codons !
Some tRNA can read more than one codon !
This is because the first base of the anti-codon (that binds to the third base of the mRNA codon) is not squeezed/ constrained as it would be in a DNA double helix and can wobble making other base pairings possible i.e.
‘ wobble base-paring ‘
Therefore a single tRNA can two recognise two different codons for the same amino acid !
Adenosine to inosine conversion at the wobble position of the anticodon in some tRNAs permit it to recognise three different codons !
Figure 6-53 Molecular Biology of the Cell (© Garland Science 2008)

MOLECULAR BIOLOGY – Protein synthesis
http://www.youtube.com/watch?v=4MRCH_J7Fhk

MOLECULAR BIOLOGY – Protein synthesis
Attachment of aminoacids to tRNAs (‘Charging’)
Each tRNA is charged by a specific enzymes that recognise both the tRNA and the amino acid - called ‘aminoacyl tRNA synthetases‘ e.g.
tryptophanyl tRNA synthetase
Charging is a two step process
Figure 6-58 Molecular Biology of the Cell (© Garland Science 2008)

MOLECULAR BIOLOGY – Protein synthesis tRNA charging
2. Transfer of the amino acid to the free 3’OH of the tRNA
Uncharged tRNA
1. Amino acid adenylation
(Aminoacyl-AMP)
Figure 6-56 Molecular Biology of the Cell (© Garland Science 2008)
Charged tRNA

MOLECULAR BIOLOGY – Protein synthesis
http://www.phschool.com/science/biology_place/biocoach/translation/addaa.html

MOLECULAR BIOLOGY – Protein synthesis
During protein synthesis tRNAs are sequentially released from their corresponding amino acids peptide bond amino (N-) terminus carboxyl (C-) terminus
What is responsible for the formation of peptide bonds within the cell ?
Figure 6-61 Molecular Biology of the Cell (© Garland Science 2008)

MOLECULAR BIOLOGY – Protein synthesis
Very large protein-RNA complexes called ‘Ribosomes’
Ribosome comprise one large and one small subunit
Ribosomes bind both the mRNA and amino acid charged tRNAs to decode the information in the mRNA into a polypeptide sequence of amino acids
Figure 6-63 Molecular Biology of the Cell (© Garland Science 2008)

MOLECULAR BIOLOGY – Protein synthesis
Ribosomal RNA (‘rRNA’) critical to ribosome function
Prokaryotic 16S rRNA rRNAs:
•
2/3 of the molecular weight for ribosome
(prokaryotes)
• form complex and defined secondary structure
• originally thought to have structural role, now known to required for most of the ribosome’s functions
•
X-ray crystallography show no proteins are proximal to catalytic site to participate in peptide bond formation
•
23S rRNA (prokaryotes) acts as a ‘peptidyl transferase
’ ribozyme
• sequence mutagenesis studies of 23S rRNA show its function is to correctly position the incoming charged tRNA to allow spontaneous formation of the peptide bond

MOLECULAR BIOLOGY – Protein synthesis
3D ribosomal structure (70S prokaryotic)
Figure 6-64 Molecular Biology of the Cell (© Garland Science 2008)
The interface between large & small s/u’s form a groove for mRNA binding and three tRNA binding sites: A (acceptor), P
(peptide) & E (exit)

MOLECULAR BIOLOGY – Protein synthesis
Correctly identifying the translation start-point in mRNA
Prokaryotic ribosomes
Translation always starts at an AUG codon (coding for methionine) called the ‘ start codon
’
How does the ribosome recognise the correct AUG as the start codon ?
Various ‘initiation factors (IFs) ’ participate in this process
Shine-Delgarno sequence
N-Formyl methionine charged tRNA is then recruited into the P-site ready for translation to start
7bp nnnnnnAGGAGGUnnnnnnn AUG nnnnnnn
UCCUCCA start codon mRNA
Enables translation of polycistronic mRNAs
16S rRNA base-pairing leads to small ribosomal s/u recognition, large s/u recruitment and formation of the
‘
70S initiation complex
’
Shine-Delgarno sequence
Variations in the S-D sequence can effect translation initiation efficiency

MOLECULAR BIOLOGY – Protein synthesis
http://www.biostudio.com/d_%20Protein%20Synthesis%20Prokaryotic.htm

MOLECULAR BIOLOGY – Protein synthesis
Eukaryotic ribosomes
The 5’ cap structure of the mRNA is recognised leading to the recruitment of the 40S small ribosome s/u and the initiator tRNA met and this initiator complex ‘ scans ’ in a 5’ to 3’ direction until the first AUG is recognised eIF3 (small 40S ribosome s/u binding) m 7 G 5’-cap eIF2 (initiator tRNA met binding)
Small 40S ribosomal s/u eIF4 (cap binding)
‘ ribosome scanning ’
‘
Eukaryotic initiation factors (eIFs)
’ facilitate the process
N.B.
the sequence context of the AUG is important
(consensus
GCCRCCAUGG) meaning some AUG‘s maybe skipped
Figure 6-72 (part 2 of 5) Molecular Biology of the Cell (© Garland Science 2008)

MOLECULAR BIOLOGY – Protein synthesis
Ribosome scanning leads to the selection of the appropriate start codon/ AUG eIF5 assisted
Ribosome now correctly placed to ‘read the correct frame in the mRNA
Figure 6-72 (part 3 of 5) Molecular Biology of the Cell (© Garland Science 2008)

MOLECULAR BIOLOGY – Protein synthesis
Elongation phase of translation
Bound tRNAs move to next site (A-P or P-E)
Charged tRNA enters A-site.
Specificity dictated by codon-anticodon base-pairing
New peptide bond formation
(between adjacent amino acids in P
& A-sites)
As next charged tRNA enters Asite the E-site occupant departs the ribosome
Ribosome
‘translocates’ along mRNA to next codon
N.B.
The A- and E-sites can never be simultaneously occupied
Figure 6-66 Molecular Biology of the Cell (© Garland Science 2008)
The elongation phase of translation is essentially similar in prokaryotes and eukaryotes involving a repetition of a series of steps

MOLECULAR BIOLOGY – Protein synthesis
The elongation phase is governed ‘elongation factors (EFs)’
Prokaryotic example used below (eukaryotes have other EFs but principle is the same)
‘ EF-Ts ’ exchanges GDP from EF-Tu for fresh GTP allowing it to recruit more charged tRNAs to the Asite
EF-Ts
GDP
Figure 6-67 (part 1 of 7) Molecular Biology of the Cell (© Garland Science 2008)
‘
EF-Tu
’ binds to charged tRNAs and delivers them to the A-site.
This requires energy from GTP hydrolysis to GDP

MOLECULAR BIOLOGY – Protein synthesis
Ribosomal TRANSLOCATION along the mRNA and the associated migration of the t-RNAs from the A- to P-site or P- to E-site also requires energy from GTP hydrolysis mediated by ‘ EF-G ’
EF-G binding causes bound tRNAs to exist partially bound to both sites (A & P or P & E) and GTP hydrolysis completes the translocation
Figure 6-67 (part 6 of 7) Molecular Biology of the Cell (© Garland Science 2008)

MOLECULAR BIOLOGY – Protein synthesis
Termination of translation
1.
No tRNAs can recognise a stop codon in the A-site. The stop codon is therefore recognised by a ‘release factor (RF)’ (either RF1 or RF2 depending on stop codon sequence)
2.
RFs activate the peptidyl-transferase of the ribosome to hydrolyse the bond between the completed polypeptide chain and the tRNA in the P-site
3.
Further RFs (RF3 and ‘Ribosome recycling factor (RRF)’ dissociate RF1/2 and the small/ large ribosomal s/u’s

MOLECULAR BIOLOGY – Protein synthesis
>1 ribosome can translate a single mRNA at a time
‘Polysome (i.e. polyribosome)’ formation in eukaryotes
The 5’ cap binding protein (eIF4) interacts with PABP (poly A-binding protein) at the 3’ end of the mRNA with translating ribosomes at approx 100bp intervals around the length of the mRNA transcript
Transmission electron micrograph
N.B.
In eukaryotes the mRNA is extensively processed in the nucleus before being exported into the cytoplasm for translation
Figure 6-76 Molecular Biology of the Cell (© Garland Science 2008)

MOLECULAR BIOLOGY – Protein synthesis
In bacteria mRNA transcription and translation are coupled in the cytoplasm !
Even as the mRNA sequence is appearing from the RNA polymerase complex it is recognised by ribosomes and immediately translated into protein !
N.B.
the mRNA transcripts are often polycistronic coding for more than one protein !

MOLECULAR BIOLOGY – Protein synthesis
Although mechanistically similar, prokaryotic and eukaryotic ribosomes are not identical
MANY ANTIBIOTICS WORK BY INHIBITING BACTERIAL PROTEIN SYNTHESIS
30S
Figure 6-79 Molecular Biology of the Cell (© Garland Science 2008)
50S

MOLECULAR BIOLOGY – Protein synthesis
Small molecule inhibitors of protein synthesis
Table 6-4 Molecular Biology of the Cell (© Garland Science 2008)

MOLECULAR BIOLOGY – Protein synthesis
http://bcs.whfreeman.com/thelifewire/content/chp12/1202003.html

MOLECULAR BIOLOGY – Protein structure & function

MOLECULAR BIOLOGY – Protein structure & function
Proteins are polymers of different amino acids joined by peptide bonds
Amino acids
Protein synthesis
Polypeptide ( i.e.
protein)
Each amino acid has a different chemical side chain and the order of these side chains in a protein sequence is what conveys its structure and functionality
Figure 3-1 (part 1 of 2) Molecular Biology of the Cell (© Garland Science 2008)

MOLECULAR BIOLOGY – Protein structure & function
Grouping the 20 amino acids by their chemical properties
Hydrophylic Hydrophobic
Learn the amino acid abbreviations and properties
Figure 3-2 Molecular Biology of the Cell (© Garland Science 2008)

MOLECULAR BIOLOGY – Protein structure & function
Types of chemical bonding contributing to protein structure formation
Non covalent bond/ interactions
N.B. Non covalent bonding can exist between any combination of the amino acid side chains and the peptide backbone
Figure 3-4 Molecular Biology of the Cell (© Garland Science 2008) covalent peptide bond covalent disulphide bond
(between two Cys side chains)

MOLECULAR BIOLOGY – Protein structure & function
Chemical bonding results in 4 levels of protein structure
Primary (1 o )
The simple order of the amino acids in the polypeptide chain
Secondary (2 o )
Interactions between the amino acid
(mostly hydrogen bonds) resulting in an array of regular sub-structures (
 helix
 strand)
Quaternary (4 o )
The relative arrangement of multiple proteins ( i.e.
tertiary structures) in a complex i.e.
sub units
Tertiary (3 o )
The overall 3D structure of a protein describing the spatial arrangement of the secondary structural elements (themselves often found in discreet motifs)

Alpha-helix
MOLECULAR BIOLOGY – Protein structure & function
Type of 2 o protein structure
A right handed coiled conformation in which the NH group of an amino acid in the peptide backbone forms a hydrogen bond (shown in yellow and pink opposite)with the CO group of an amino acid 4 residues earlier
Beta-strand (leading to beta sheets)
The polypeptide chain exists in a stretched conformation and peptide backbone hydrogen bonds form between the NH and CO groups (light blue) of amino acids in different strands
As the polypeptide chain has polarity (i.e. an N- and Cterminus) the two strands can run PARALLEL or ANTI-
PARALLEL to each other
The arrangement of beta strands forms a beta sheet

MOLECULAR BIOLOGY – Protein structure & function
2 o structural elements within a solved protein 3 o structure (e.g. using Xray crystallography) are often represented by ‘ribbon diagrams
’
Alphahelix
Anti-parallel beta-sheet
Parallel beta-sheet e.g.
dihydrofolate reductase

MOLECULAR BIOLOGY – Protein structure & function
Newly translated proteins must ‘fold’ to attain functional structure
Chemical properties of the amino acid side chains and primary protein structure contribute to the spontaneous folding pattern
Figure 3-5 Molecular Biology of the Cell (© Garland Science 2008)

MOLECULAR BIOLOGY – Protein structure & function
Protein folding and forming a functional structure is complex !
Correct incorporation of essential ‘ cofactors
’ during polypeptide chain folding e.g.
metal ions in enzymes, rRNAs in ribosomes
Addition of ‘ post-translational ’ covalent modifications required for protein activity or recruitment of other proteins e.g.
phosphorylation
Successful assembly of multi-protein complexes required to attain functionality e.g.
ribosome assembly
Figure 6-82 Molecular Biology of the Cell (© Garland Science 2008)

MOLECULAR BIOLOGY – Protein structure & function
The cell’s cytoplasm is ‘molecularly crowded’
CYTOPLASM IS TIGHTLY PACKED
WITH MOLECULES
Cytoplasm
PROBLEM: How do newly produced polypeptide chains fold appropriately without forming aggregates with the ‘molecular crowd’ of other proteins in the cytoplasm?

MOLECULAR BIOLOGY – Protein structure & function
Chaperonins/ chaperons needed for proper folding
Chaperonins/ Chaperons:
• proteins within the cell that assist with appropriate folding of proteins
• their role is to prevent misfolding rather than actively direct correct folding
• can act to delay any folding (e.g. as the nascent polypeptide chain emerges from the ribosome)
• can also ‘rescue’ misfolded proteins to the correct folding conformation
Chaperonins/ chaperons exist to ensure that nothing inappropriate occurs ! . . . (in a protein folding sense).

MOLECULAR BIOLOGY – Protein structure & function
e.g. Heat Shock Protein 70 (Hsp70)
1. Hsp70ATP able to loosely bind hydrophobic patches of amino acids as they emerge from the ribosome
3. Hsp70ADP tightly associates with unfolded protein and protects it from aggregating
5. Protein spontaneously folds into correct confirmation
2. Peptide binding induces intrinsic ATPase activity in
HSP70
4. Nucleotide exchange factors eventually replace the ADP with
ATP and HSP70 releases the unfolded protein
6. A small percentage of protein incorrectly folds
The expression of Hsps (heat shock proteins) increases as temperature increases because folded proteins are more likely to unfold/ denature at higher temperatures
Figure 6-86 Molecular Biology of the Cell (© Garland Science 2008)

MOLECULAR BIOLOGY – Protein structure & function e.g.
GroEL/ Hsp60 ‘rescues’ misfolded proteins
Multi-subunit complex
‘cocktail shaker’
1. Misfolded proteins with exposed hydrophobic regions bind hydrophobic regions in the neck of the GroEL 3. Hydrolysis of the bound ATP (plus binding of additional ATP) releases the GroES cap and the correctly folded protein
2. The binding of the GroES cap and
ATP cause conformational change that releases the misfolded protein into the lumen where it can fold, sequestered from the cytoplasm
4. Another misfolded protein binds the opposite side of the GroEL complex
Figure 6-87 Molecular Biology of the Cell (© Garland Science 2008)

MOLECULAR BIOLOGY – Protein structure & function
http://bcs.whfreeman.com/lodish5e/content/cat_010/03010-01.htm?v=chapter&i=03010.01&s=03000&n=00010&o =

MOLECULAR BIOLOGY – Protein structure & function
Very large 2MDa protein nuclear & cytoplasmic complex (26S)
20S core particle is formed from stacked heptameric rings creating a hollow cavity for safe protein digestion
19S regulatory particle
Structural/ regulatory
 subunits
20S core particle
Proteolytically active
 subunits
26S proteasome (cryo-electron microscopy)
How are condemned proteins targeted to the proteasome ?

MOLECULAR BIOLOGY – Protein structure & function
‘Ubiquitin’ - 76 amino acid highly conserved polypeptide found in all cells that when attached to a condemned protein in multiple copies targets it the proteasome ( i.e.
a destruction signal)
Ubiquitination of condemned proteins requires three enzymes:
1) E1 ubiquitin activating enzyme hydrolyses ATP to attaches itself to and thus activate ubiquitin
2) E2 ubiquitin conjugating enzyme recognises the E1-ubquitin complex and transfers the complex to itself
3) E3 ubiquitin ligase enzyme binds the condemned protein substrate and an E2-ubiquitin complex thus allowing E2 to transfer the ubiquitin to the protein to be destroyed. This process is repeated multiple times.
Poly-ubiquitinated proteins are then targeted to the proteasome where ubiquitin is recognised by binding sites on the 19S particle and removed for recycling. Energy from ATP hydrolysis unfolds and feeds the protein into the catalytic core for destruction into amino acids and peptides

MOLECULAR BIOLOGY – Protein structure & function
http://www.sinauer.com/cooper5e/animation0802.html

MOLECULAR BIOLOGY – Protein structure & function
Protein folding timeline
Figure 6-88 Molecular Biology of the Cell (© Garland Science 2008)

MOLECULAR BIOLOGY – Protein structure & function
Proteins not just targeted for destruction !
Specific sequences of amino acids can direct proteins to the correct sub-cellular location for their function (e.g. the nucleus, mitochondria etc.) e.g.
‘ signal sequences
’ targeting proteins for cell export
2) Translation is stalled and the SRP targets the ribosome to a membrane ‘translocation complex’
1) a 20 amino acid
N-terminal
‘signal sequence
’ emerging from the ribosome is bound by the
‘signal recognition particle
(SRP)
’
3) SRP dissociation restarts translation and the growing polypeptide chain is past across the membrane ‘co-translationally’
Highly conserved between eukaryotes and prokaryotes
(eukaryotic exported proteins pass across the ER membrane rather than the plasma membrane)
4) The signal sequence is enzymatically removed and the protein folds

MOLECULAR BIOLOGY – Protein structure & function
e.g.
http://www.sinauer.com/cooper5e/animation1001.html

MOLECULAR BIOLOGY – Protein structure & function
Proteins perform many diverse functions
Information processing proteins receptors, signalling Structural proteins
Cytoskeleton
Extracellualr matrix
Enzymes
Mechanical proteins actin, myosin
Binding proteins transport, storage
Proteins are therefore subject to tight regulation to control these functions

MOLECULAR BIOLOGY – Protein structure & function
Enzymes comprise a large family of proteins
Enzymes and the reactions that they catalyse are central to regulating the activity and function of other proteins i.e.
they are important regulators
Table 3-1 Molecular Biology of the Cell (© Garland Science 2008)

MOLECULAR BIOLOGY – Protein structure & function
Enzymes can modify proteins by the addition of molecular moieties i.e. ‘post-translational modifications’
Although the genetic code specifies for the incorporation of only 20 amino acids into proteins, these can be extensively modified to confer differing functionalities by:
Phosphorylation
N-acetylation
S-prenylation
GPI-anchoring
S-Nitrosylation
Glycosylation
N-myristoylation
Sumoylation
Lipidation
Lipidation
Methylation
Deamination
S-pamitoylation
Ubiquitination

MOLECULAR BIOLOGY – Protein structure & function
There exists huge potential for complex post-translational regulation of protein function e.g.
multiple possible combinations of post-translational modification of the transcription factor p53.
Figure 3-81a Molecular Biology of the Cell (© Garland Science 2008)

MOLECULAR BIOLOGY – Protein structure & function
Post-translational protein modifications help increase the possible variety in the products of a single gene
Such increased variety in the proteome allows for greater regulation of protein function and output !

MOLECULAR BIOLOGY – Protein structure & function
e.g. phosphorylation status of an enzyme can dictate its activity
The interplay between the kinase (adding phosphate) and the phosphatase (removing phosphate) regulates whether enzyme ‘ x
’ is active or not
Figure 3-64 Molecular Biology of the Cell (© Garland Science 2008)

MOLECULAR BIOLOGY – Protein structure & function
Glycogen metabolism in the liver is regulated by phosphorylation
A signal to stop storing glucose as glycogen and to start mobilising it is processed through protein kinase A.
Figure 3-73 Molecular Biology of the Cell (© Garland Science 2008)
Phosphorylation of glycogen synthase inactivates glycogen production
Whereas phosphorylation of glycogen phoshorylase causes it’s activation (via an intermediate kinase) leading to glycogen breakdown and glucose production

MOLECULAR BIOLOGY – Protein structure & function
Various signals input into the establishment and hence readout of this ‘code’
Figure 3-81c Molecular Biology of the Cell (© Garland Science 2008)