MOLECULAR BIOLOGY – Protein synthesis PROTEIN SYNTHESIS TRANSLATION.

MOLECULAR BIOLOGY – Protein synthesis

PROTEIN SYNTHESIS

TRANSLATION

Figure 6-50 Molecular Biology of the Cell (© Garland Science 2008)


The Genetic Code

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)



Genetic code can be read in 3 ways depending upon where you start!

+1 frameshift

+2 frameshift

The genetic information encoded in each reading frame is different

DIF

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Transfer RNAs (tRNA) act as adapters between the mRNA and protein synthesising machinery (‘ribosomes’)

How is the mRNA genetic code read during protein synthesis?

tRNA triplet nucleotide sequences that are complementary to mRNA codons, called ‘anticodons’, form specific base-pairs with the mRNA codons

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

Some tRNA can read more than one codon !

Adenosine to inosine conversion at the wobble position of the anticodon in some

tRNAs permit it to recognise three different codons !


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‘


The minimum set of required tRNAs is 31 but there are 61 possible amino acid coding codons !

Therefore a single tRNA can two recognise two different codons for the same amino acid !


tRNA structure summary video/ tutorial

http://www.youtube.com/watch?v=4MRCH_J7Fhk

http://www.youtube.com/watch?v=4MRCH_J7Fhk



Attachment of amino-acids 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



(Aminoacyl-AMP)

tRNA charging

Uncharged tRNA

Charged tRNA

2. Transfer of the amino acid to the free 3’OH of

the tRNA

1. Amino acid adenylation


http://www.phschool.com/science/biology_place/biocoach/translation/addaa.html

tRNA amino acid charging video/ tutorial

http://www.phschool.com/science/biology_place/biocoach/translation/addaa.html


amino (N-) terminus carboxyl (C-) terminuspeptide bond


During protein synthesis tRNAs are sequentially released from their corresponding amino acids

What is responsible for the formation of peptide bonds within the cell ?



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

Prokaryotic 16S rRNA


Ribosomal RNA (‘rRNA’) critical to ribosome function

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



3D ribosomal structure (70S prokaryotic)

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)

Prokaryotic ribosomes

nnnnnnAGGAGGUnnnnnnnAUGnnnnnnn UCCUCCA

Shine-Delgarno sequence

start codon

16S rRNA base-pairing leads to small ribosomal s/u recognition, large s/u

recruitment and formation of the ‘70S initiation complex’


Correctly identifying the translation start-point in mRNA

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 ?

7bp

Shine-Delgarno sequence

Enables translation of polycistronic mRNAs

N-Formyl methionine charged tRNA is then recruited into the P-site ready for translation to start

mRNA

Various ‘initiation

factors (IFs)’ participate in this

process

Variations in the S-D sequence can effect translation initiation efficiency


Prokaryotic translation summary video/ tutorial (including

inititation)

http://www.biostudio.com/d_%20Protein%20Synthesis%20Prokaryotic.htm

http://www.biostudio.com/d_%20Protein%20Synthesis%20Prokaryotic.htm

Figure 6-72 (part 2 of 5) Molecular Biology of the Cell (© Garland Science 2008)


N.B. the sequence context of the AUG is important

(consensus GCCRCCAUGG) meaning

some AUG‘s maybe skipped

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 tRNAmet and this initiator complex ‘scans’ in a 5’ to 3’ direction until

the first AUG is recognised

m7G 5’-cap

Small 40S ribosomal s/u

‘ribosome scanning’

eIF4 (cap binding)

eIF2 (initiator tRNAmet binding)

eIF3 (small 40S ribosome s/u binding)

‘Eukaryotic initiation factors (eIFs)’ facilitate

the process



Ribosome now correctly placed to ‘read the correct frame in the mRNA

eIF5 assisted

Ribosome scanning leads to the selection of the appropriate start codon/ AUG



The elongation phase of translation is essentially similar in prokaryotes

and eukaryotes involving a repetition of a series of steps

Elongation phase of translation

Charged tRNA enters A-site.

Specificity dictated by

codon-anticodon base-pairing

New peptide bond formation

(between adjacent amino acids in P

& A-sites)

Ribosome ‘translocates’ along mRNA to

next codon

Bound tRNAs move to next site (A-P or P-E)

As next charged tRNA enters A-site the E-site occupant

departs the ribosome

N.B. The A- and E-sites can never be simultaneously occupied



The elongation phase is governed ‘elongation factors (EFs)’

Prokaryotic example used below (eukaryotes have other EFs but principle is the same)

‘EF-Tu’ binds to charged tRNAs and delivers them to the A-site. This requires energy from GTP

hydrolysis to GDP

GDP

EF-Ts

‘EF-Ts’ exchanges GDP from EF-Tu for fresh GTP allowing it to recruit more charged tRNAs to the A-

site


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


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)


Termination of translation

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


‘Polysome (i.e. polyribosome)’ formation in eukaryotes


>1 ribosome can translate a single mRNA at a time

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


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 !


MANY ANTIBIOTICS WORK BY INHIBITING BACTERIAL PROTEIN SYNTHESIS


Although mechanistically similar, prokaryotic and eukaryotic ribosomes are not identical

30S 50S

Table 6-4 Molecular Biology of the Cell (© Garland Science 2008)


Small molecule inhibitors of protein synthesis


http://bcs.whfreeman.com/thelifewire/content/chp12/1202003.html

Translation summary video/ tutorial






PROTEIN STRUCTURE AND FUNCTIONS

MOLECULAR BIOLOGY – Protein structure & function



Proteins are polymers of different amino acids joined by peptide bonds

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

Amino acids

Polypeptide (i.e. protein)

Protein synthesis


Hydrophylic Hydrophobic


Grouping the 20 amino acids by their chemical properties

Learn the amino acid abbreviations and properties



Types of chemical bonding contributing to protein structure formation

covalent disulphide bond(between two Cys side chains)

Non covalent bond/ interactions

covalent peptide bond

N.B. Non covalent bonding can exist between any combination of the amino acid side chains

and the peptide backbone


Chemical bonding results in 4 levels of protein structure

Primary (1o)

Secondary (2o)

Tertiary (3o)

Quaternary (4o)

The simple order of the amino acids in the polypeptide chain

Interactions between the amino acid (mostly hydrogen bonds) resulting in an

array of regular sub-structures ( helix strand)

The overall 3D structure of a protein describing the spatial arrangement of the secondary structural

elements (themselves often found in discreet

motifs)

The relative arrangement of multiple proteins (i.e. tertiary structures) in a complex i.e. sub units

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Type of 2o protein structure

Alpha-helixA 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 C-terminus) the two strands can run PARALLEL or ANTI-

PARALLEL to each other

The arrangement of beta strands forms a beta sheet


2o structural elements within a solved protein 3o structure (e.g. using X-ray crystallography) are often represented by ‘ribbon

diagrams’

Alpha-helix

Anti-parallel beta-sheet

e.g. dihydrofolate reductase

Parallel beta-sheet



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



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


The cell’s cytoplasm is ‘molecularly crowded’

Cytoplasm

CYTOPLASM IS TIGHTLY PACKED WITH MOLECULES

PROBLEM: How do newly produced polypeptide chains fold appropriately without forming aggregates

with the ‘molecular crowd’ of other proteins in the cytoplasm?


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).



e.g. Heat Shock Protein 70 (Hsp70)

The expression of Hsps (heat shock proteins) increases as temperature increases because folded proteins are more likely to unfold/ denature at higher temperatures

1. Hsp70-ATP able to loosely bind hydrophobic patches of amino acids as they emerge from the

ribosome

2. Peptide binding induces intrinsic ATPase activity in

HSP70

3. Hsp70-ADP tightly associates with unfolded protein and

protects it from aggregating

4. Nucleotide exchange factors eventually replace the ADP with

ATP and HSP70 releases the unfolded protein

5. Protein spontaneously folds into correct confirmation

6. A small percentage of protein incorrectly folds



e.g. GroEL/ Hsp60 ‘rescues’ misfolded proteins

1. Misfolded proteins with exposed hydrophobic regions bind hydrophobic

regions in the neck of the GroEL

Multi-subunit complex ‘cocktail shaker’

2. The binding of the GroES cap andATP cause conformational change that releases the misfolded protein into the lumen where it can fold, sequestered from the

cytoplasm

3. Hydrolysis of the bound ATP (plus binding of additional ATP) releases the GroES cap and the

correctly folded protein

4. Another misfolded protein binds the opposite side of the GroEL complex


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

Chaperone mediated protein folding summary video/ tutorial

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

















Unneeded and misfolded proteins are degraded by the eukaryotic ‘proteasome’

Very large 2MDa protein nuclear & cytoplasmic complex (26S)

19S regulatory particle

20S core particle

26S proteasome (cryo-electron microscopy)

20S core particle is formed from stacked heptameric rings creating a hollow cavity

for safe protein digestion

Structural/ regulatory subunits

Proteolytically active

subunits

How are condemned proteins targeted to the proteasome ?

Protein are targetted to the protesome by ‘poly-ubiquitination’


Ubiquitination of condemned proteins requires three enzymes:

‘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)

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


http://www.sinauer.com/cooper5e/animation0802.html

Ubiquitin/ proteosome mediated protein degradation summary

video/ tutorial




Protein folding timeline


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

1) a 20 amino acid N-terminal ‘signal sequence’ emerging from the ribosome is bound by the ‘signal recognition particle (SRP)’

2) Translation is stalled and the SRP targets the ribosome to a membrane ‘translocation complex’

3) SRP dissociation restarts translation and the growing polypeptide chain is past across the membrane ‘co-translationally’

4) The signal sequence is enzymatically removed and the protein folds

Highly conserved between eukaryotes and prokaryotes

(eukaryotic exported proteins pass across the ER membrane rather than the plasma

membrane)


Protein targeting e.g. signal recognition sequences and cell export summary video/ tutorial



Structural proteinsCytoskeletonExtracellualr matrix

Mechanical proteinsactin, myosin

Enzymes

Binding proteinstransport, storage

Information processing proteinsreceptors, signalling


Proteins perform many diverse functions

Proteins are therefore subject to tight regulation to control these functions

Table 3-1 Molecular Biology of the Cell (© Garland Science 2008)


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


Enzymes can modify proteins by the addition of molecular moieties i.e. ‘post-translational modifications’

Phosphorylation GlycosylationMethylation

N-acetylation N-myristoylation Deamination

S-prenylation Sumoylation S-pamitoylation

GPI-anchoring Lipidation Ubiquitination

S-Nitrosylation Lipidation

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:

Figure 3-81a Molecular Biology of the Cell (© Garland Science 2008)


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.


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 !



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



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.

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

Figure 3-81c Molecular Biology of the Cell (© Garland Science 2008)


Various signals input into the establishment and hence readout of this ‘code’

MOLECULAR BIOLOGY – Protein synthesis PROTEIN SYNTHESIS TRANSLATION.

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