MOLECULAR BIOLOGY – Protein synthesis

MOLECULAR BIOLOGY – Protein synthesis
TRANSLATION

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

Genetic code can be read in 3 ways depending upon where you start!
MOLECULAR BIOLOGY – Protein synthesis Genetic code can be read in 3 ways depending upon where you start! The genetic information encoded in each reading frame is different DIFFERENT READING FRAMES OF mRNA THE SAME SEQUENCE +1 frameshift +2 frameshift Figure Molecular Biology of the Cell (© Garland Science 2008)

How is the mRNA genetic code read during protein synthesis?
MOLECULAR BIOLOGY – Protein synthesis How is the mRNA genetic code read during protein synthesis? Figure Molecular Biology of the Cell (© Garland Science 2008) 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

The minimum set of required tRNAs is 31 but there are 61 possible amino acid coding codons ! Figure Molecular Biology of the Cell (© Garland Science 2008) 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‘ Some tRNA can read more than one codon ! 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 !

tRNA structure summary video/ tutorial

Attachment of amino-acids to tRNAs (‘Charging’)
MOLECULAR BIOLOGY – Protein synthesis 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 Figure Molecular Biology of the Cell (© Garland Science 2008)

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

tRNA amino acid charging video/ tutorial

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

Very large protein-RNA complexes called ‘Ribosomes’
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 Molecular Biology of the Cell (© Garland Science 2008)

Ribosomal RNA (‘rRNA’) critical to ribosome function
MOLECULAR BIOLOGY – Protein synthesis 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 Prokaryotic 16S rRNA

3D ribosomal structure (70S prokaryotic)
MOLECULAR BIOLOGY – Protein synthesis 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) Figure Molecular Biology of the Cell (© Garland Science 2008)

Correctly identifying the translation start-point in mRNA
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 ? Shine-Delgarno sequence N-Formyl methionine charged tRNA is then recruited into the P-site ready for translation to start 7bp nnnnnnAGGAGGUnnnnnnnAUGnnnnnnn UCCUCCA mRNA start codon Various ‘initiation factors (IFs)’ participate in this process 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 Enables translation of polycistronic mRNAs Variations in the S-D sequence can effect translation initiation efficiency

Prokaryotic translation summary video/ tutorial (including inititation)

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 eIF4 (cap binding) eIF2 (initiator tRNAmet binding) eIF3 (small 40S ribosome s/u binding) ‘Eukaryotic initiation factors (eIFs)’ facilitate the process m7G 5’-cap Small 40S ribosomal s/u ‘ribosome scanning’ 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)

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

Elongation phase of translation
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 A-site the E-site occupant departs the ribosome Ribosome ‘translocates’ along mRNA to next codon The elongation phase of translation is essentially similar in prokaryotes and eukaryotes involving a repetition of a series of steps N.B. The A- and E-sites can never be simultaneously occupied Figure Molecular Biology of the Cell (© Garland Science 2008)

The elongation phase is governed ‘elongation factors (EFs)’
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 ‘EF-Ts’ exchanges GDP from EF-Tu for fresh GTP allowing it to recruit more charged tRNAs to the A-site GDP ‘EF-Tu’ binds to charged tRNAs and delivers them to the A-site. This requires energy from GTP hydrolysis to GDP Figure 6-67 (part 1 of 7) Molecular Biology of the Cell (© Garland Science 2008)

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)

Termination of translation
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

>1 ribosome can translate a single mRNA at a time
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 Molecular Biology of the Cell (© Garland Science 2008)

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 !

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

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

Translation summary video/ tutorial

MOLECULAR BIOLOGY – Protein structure & function
PROTEIN STRUCTURE AND FUNCTIONS

Proteins are polymers of different amino acids joined by peptide bonds
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)

Grouping the 20 amino acids by their chemical properties
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)

Types of chemical bonding contributing to protein structure formation
MOLECULAR BIOLOGY – Protein structure & function Types of chemical bonding contributing to protein structure formation Non covalent bond/ interactions covalent disulphide bond (between two Cys side chains) covalent peptide bond 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)

Chemical bonding results in 4 levels of protein structure
MOLECULAR BIOLOGY – Protein structure & function Chemical bonding results in 4 levels of protein structure Covalent peptide bonding 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 Amino acid side chain interactions Primary structure - the amino acid sequence of the peptide chains. Secondary structure - highly regular sub-structures (alpha helix and strands of beta sheet) which are locally defined, meaning that there can be many different secondary motifs present in one single protein molecule. Tertiary structure - Three-dimensional structure of a single protein molecule; a spatial arrangement of the secondary structures. Quaternary structure - complex of several protein molecules or polypeptide chains, usually called protein subunits in this context, which function as part of the larger assembly or protein complex.

Type of 2o protein structure
MOLECULAR BIOLOGY – Protein structure & function Type of 2o protein structure Alpha-helix 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 C-terminus) the two strands can run PARALLEL or ANTI-PARALLEL to each other The arrangement of beta strands forms a beta sheet Primary structure - the amino acid sequence of the peptide chains. Secondary structure - highly regular sub-structures (alpha helix and strands of beta sheet) which are locally defined, meaning that there can be many different secondary motifs present in one single protein molecule. Tertiary structure - Three-dimensional structure of a single protein molecule; a spatial arrangement of the secondary structures. Quaternary structure - complex of several protein molecules or polypeptide chains, usually called protein subunits in this context, which function as part of the larger assembly or protein complex.

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 Primary structure - the amino acid sequence of the peptide chains. Secondary structure - highly regular sub-structures (alpha helix and strands of beta sheet) which are locally defined, meaning that there can be many different secondary motifs present in one single protein molecule. Tertiary structure - Three-dimensional structure of a single protein molecule; a spatial arrangement of the secondary structures. Quaternary structure - complex of several protein molecules or polypeptide chains, usually called protein subunits in this context, which function as part of the larger assembly or protein complex. Parallel beta-sheet e.g. dihydrofolate reductase

Newly translated proteins must ‘fold’ to attain functional structure
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)

Protein folding and forming a functional structure is complex !
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 Molecular Biology of the Cell (© Garland Science 2008)

CYTOPLASM IS TIGHTLY PACKED WITH MOLECULES
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?

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)
MOLECULAR BIOLOGY – Protein structure & function e.g. Heat Shock Protein 70 (Hsp70) 3. Hsp70-ADP tightly associates with unfolded protein and protects it from aggregating 1. Hsp70-ATP able to loosely bind hydrophobic patches of amino acids as they emerge from the ribosome 5. Protein spontaneously folds into correct confirmation 4. Nucleotide exchange factors eventually replace the ADP with ATP and HSP70 releases the unfolded protein 2. Peptide binding induces intrinsic ATPase activity in HSP70 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 Molecular Biology of the Cell (© Garland Science 2008)

Multi-subunit complex ‘cocktail shaker’
MOLECULAR BIOLOGY – Protein structure & function Multi-subunit complex ‘cocktail shaker’ e.g. GroEL/ Hsp60 ‘rescues’ misfolded proteins 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 Molecular Biology of the Cell (© Garland Science 2008)

Chaperone mediated protein folding summary video/ tutorial
MOLECULAR BIOLOGY – Protein structure & function Chaperone mediated protein folding summary video/ tutorial

Unneeded and misfolded proteins are degraded by the eukaryotic ‘proteasome’ 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 Proteolytically active subunits 20S core particle How are condemned proteins targeted to the proteasome ? 26S proteasome (cryo-electron microscopy)

Protein are targetted to the protesome by ‘poly-ubiquitination’
MOLECULAR BIOLOGY – Protein structure & function Protein are targetted to the protesome by ‘poly-ubiquitination’ ‘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

Ubiquitin/ proteosome mediated protein degradation summary video/ tutorial

Highly conserved between eukaryotes and prokaryotes
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

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

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

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)

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 Glycosylation Methylation N-acetylation N-myristoylation Deamination S-prenylation Sumoylation S-pamitoylation GPI-anchoring Lipidation Ubiquitination S-Nitrosylation Lipidation

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)

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
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 Molecular Biology of the Cell (© Garland Science 2008)

Glycogen metabolism in the liver is regulated by phosphorylation
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. 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 Molecular Biology of the Cell (© Garland Science 2008)

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

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