Principles of Cell Biology (BIOL2060)

Department of Biology
Memorial University of Newfoundland

Protein Synthesis

Translation is the first & most important part of protein synthesis.
Translation is a well-conserved process among prokaryotes and eukaryotes.
Additional processing and assembly is often required to modify the proteins.
Ribosomes catalyze the joining of the amino acid monomers directed by the mRNA sequence.
Amino-acyl tRNA synthetases attach amino acids to the appropriate tRNAs.
The amino-acyl tRNA act as adaptors in the translation of the nucleic acid sequence of the mRNA into the amino acid sequence of the protein.

Translation occurs in three stages...
1) In initiation, the components of the translational apparatus come together with the mRNA molecule (A tRNA carrying the first amino acid binds to the start codon).
2) In elongation, amino acids are brought to the mRNA as amino-acyl tRNAs and are added one at a time to a growing polypeptide chain.
3) In termination, a stop codon in the mRNA is recognized by a protein release factor and the translational apparatus comes apart to release a completed polypeptide.

The Tools of Translation

The model of ribosome structure shows the A (aminoacyl) and P (peptidyl) sites as cavities on the ribosome where charged tRNA (carrying an amino acid) molecules bind during polypeptide synthesis.
The recently postulated E (exit) site is the site from which discharged tRNAs leave the ribosome.
The mRNA-binding site binds a sequence near the 5 prime end of the mRNA, placing the mRNA in the proper position for the translation of its first codon
The binding sites are all located at or near the interface between the large and small subunits.

tRNA molecules contain...
1) three major loops,
2) four base-paired regions,
3) an anticodon triplet and
4) a 3 prime terminal sequence of CCA (where the appropriate amino acid can be attached by an ester bond).
During maturation of the tRNA molecule a number of nucleotides are modified in tRNA specific ways.
The modified nucleotides in the tRNA structure are inosine (I), methylinosine (mI), dihydrouridine (D), ribothymidine (T), pseudouridine (¥) and methylguanosine (Gm).
The 3D structure of tRNA molecules is similar to a hockey stick (the L shaped tertiary structure of tRNA) which has the amino acid attachment site is at one end (tip of the handle) and the anticodon at the other (blade of the stick).

Twenty different aminoacyl-tRNA synthetases link amino acids to the correct tRNAs.
Some recognize only one tRNA, some recognize a few because of the redundancy in the genetic code.
Although there are 61 possible codons, there are far fewer tRNAs.
A number of codons that encode the same amino acid differ only in the third position of the codon.
A slight shift or "wobble" in the position of the base guanine in a tRNA anticodon would permit it to pair with uracil instead of its normal complementary base (cytosine).
The base pairs permitted at the third position of a codon by the wobble hypothesis are as follows:
Bases recognized in codon
(third position only)  Base in anticodon
A or G  U
C or U  G
U, C or A  I (inosine)

In two chemical steps, aminoacyl-tRNA synthetases catalyzes the formation of an ester bond between the carboxyl group of an amino acid and the 3 prime hydroxyl (OH) group of the appropriate tRNA.
Step 1) The amino acid and a molecule of ATP enter the active site of the enzyme.
            The ATP loses pyrophosphate and the resulting AMP bonds covalently to the amino acid.
            The pyrophosphate is hydrolyzed into two phosphate groups.
Step 2) The tRNA covalently bonds to the amino acid to displace the AMP and the aminoacyl tRNA is then released from the enzyme.

The Process of Translation

Messenger RNA brings polypeptide-coding information to the ribosome.
A prokaryotic mRNA molecule encoding a single polypeptide has 5 prime non-coding leader sequence.
The leader contains a ribosome binding site (or Shine-Dalgarno sequence.
The rest of the mRNA contains a coding sequence that starts with an AUG start codon and ends with a stop codon (UAA, UAG or UGA) and a 3 prime non-coding trailer sequence.
A polycistronic prokaryotic mRNA would generally have a set of these features for each gene.
A eukaryotic mRNA molecule has, in addition to the above, a 5 prime cap and a 3 prime poly(A) tail.
One important difference is that eukaryotic mRNAs lack a ribosome binding site (SD site).
A number of protein factors are required for the initiation, elongation and termination of translation.
N-formylmethionine (fMet) is the modified amino acid with which every polypeptide is initiated in prokaryotes.

The formation of the 70S translation initiation complex (in prokaryotes) occurs in three steps.
1) Three initiation factors (IF) and GTP bind to the small ribosomal subunit.
2) The initiator aminoacyl tRNA and mRNA are attached.
The mRNA-binding site is composed, at least in part, of a portion of the 16S rRNA of the small ribosomal subunit.
The 3 prime end of the 16S rRNA bears a pyrimidine-rich stretch that base pairs with the Shine-Dalgarno sequence of the mRNA (in prokaryotes).
3) The large ribosomal subunit joins the complex.
The resulting 70S initiation complex has fMet-tRNAfMet residing in the ribosome's P site.
In eukaryotes, translation includes a different set of initiation factors (eIFs), a slightly different assembly pathway and a non-formylated tRNA met.
eIF2 binds the initiator tRNA met before the small ribosomal subunit.
This complex can attach to the 5 prime cap structure of the mRNA.
As there is no Shine-Dalgarno sequence, the ribosome begins translation at a AUG that is located within the Kozak consensus sequence (often a good match to CAAAAUG).

Chain elongation during protein synthesis requires the presence of a peptidyl tRNA or, in the first elongation cycle, an fMet-tRNAfMet at the peptidyl (P) site.
1) Elongation begins with the binding of the second aminoacyl tRNA at the ribosomal aminoacyl (A) site.
The tRNA is escorted to the A site by the elongation factor EF-Tu, which also carries two bound GTPs.
As the tRNA binds, the GTPs are hydrolyzed and EF-Tu is released. EF-Ts helps recycle the EF-Tu.
2) A peptide bond is formed between the carboxyl group of the terminal amino acid (or fMet in the first cycle) at the P site and the amino group of the newly arrived amino acid at the A site.
This reaction is catalyzed by the peptidyl transferase activity of the 23S rRNA molecule in the large ribosomal subunit.
3) After EF-G-GTP binds to the ribosome and GTP is hydrolyzed, the tRNA carrying the elongated polypeptide translocates from the A site to the P site.
The discharged tRNA moves from the P site to the E (exit) site and leaves the ribosome.
As the peptidyl tRNA translocates, it takes the mRNA along with it.
Consequently, the next mRNA codon is moved into the A site, which is open for the next aminoacyl tRNA.
These events are repeated for each additional amino acid.

Termination of protein synthesis depends on release factors that recognize the three stop codons.
When a stop codon (UAG, UAA, or UGA) arrives at the A site, it is recognized and bound by a protein release factor.
This protein causes the polypeptide to be transferred to a molecule of water to cause its release from the tRNA and the dissociation of the other components of the elongation complex.

Proteins have to be folded into the proper three dimensional conformation to work properly.
A number of diseases, including Alzheimer's disease, may be considered to be protein-folding diseases.
Sometimes the primary sequence of amino acids is sufficient to spontaneously direct the folding of proteins into their proper shape.
However, often newly-made proteins require the help of molecular chaperones to attain their final shape.
The heatshock proteins, Hsp70 and Hsp60, are molecular chaperones.
Heat-denatured proteins can be renatured through the activity of molecular chaperones and heatshock proteins are made during times of stress.
Prion diseases, such as "mad cow" disease, may "self-propagate" based upon a misfolded protein that can, in turn, misfold other versions of the same protein.

After the amino chain is made, many proteins undergo posttranslational processing (including removal of stretches of amino acids).
1)  In prokaryotes, the N-formyl group is always removed in the mature protein and often the methionine and, sometimes, a number of N-terminal amino acids are cleaved away from the final protein product.
2)  The protein hormone insulin provides an example of posttranslational processing.
Proinsulin is converted to the active hormone by the enzymatic removal of a long internal section of polypeptide.
The two remaining chains continue to be covalently connected by disulfide
bonds connecting cysteine residues in insulin.
3) Recently discovered, the process of protein splicing (analagous to RNA splicing) removes inteins and splices the exteins together to make a mature protein.

Protein Targeting and Sorting

Synthesis of all polypeptides encoded by nuclear genes begins in the cytosol.
The large and small ribosomal subunits associate with each other and with the 5 prime end of an mRNA molecule, forming a functional ribosome that starts making the polypeptide.
When the polypeptide is about 30 amino acids long, it enters one of two alternative pathways.
1) In cotranslational import, if the newly forming polypeptide is destined for any of the compartments of the endomembrane system, it becomes associated with the ER membrane and is transferred across the membrane into the lumen (cisternal space) of the ER as synthesis continues.
The completed polypeptide then either remains in the ER or is transported via various vesicles and the Golgi complex to another final destination.
Integral membrane proteins are inserted into the ER membrane as they are made, rather than into the lumen.

2)  If the polypeptide is destined for the cytosol or for import into the nucleus, mitochondria, chloroplasts, or peroxisomes, its synthesis continues in the cytosol.
When the polypeptide is complete, it is released from the ribosome and either remains in the cytosol or is transported into the appropriate organelle by posttranslational import.

Polypeptide uptake by the nucleus occurs via the nuclear pores, using a mechanism different from that involved in posttranslational uptake by other organelles.

In cotranslational import, proteins to be targeted to the endoplasmic reticulum initially have an N-terminal peptide, the ER signal sequence, translated by a cytosolic ribosome.
The ER signal sequence is bound by a signal-recognition particle (SRP), a ribonucleoprotein complex composed of 6 peptides and a 300 nucleotide RNA molecule.
The SRP binds to the SRP receptor to dock the ribosome on the ER membrane.
When the SRP receptor binds GTP, the nascent polypeptide enters the pore.
The SRP is released with hydrolysis of the GTP.
The growing polypeptide translocates through a hydrophilic pore created by one or more membrane proteins called the translocon.
The most recent evidence suggests that the ribosome fits tightly across the cytoplasmic side of the pore and that the ER-lumen side is somehow closed off until the polypeptide is about 70 amino acids long.
When the polypepide is complete, the signal peptidase cleave the signal to release the protein into the ER lumen while retaining the signal peptide, for a time, in the membrane.
Afterwards the ribosome is released and the pore closes completely.

In the endoplasmic reticulum, folding of the newly-made proteins may also require molecular chaperones and other proteins involved in protein folding.
Bip (binding protein), a member of the Hsp70 chaperone family, briefly binds to and stabilizes hydrophobic regions of proteins (especially rich in Trp, Phe, Leu) allowing proper folding instead of aggregation with other inmature proteins.
Protein disulfide isomerase catalyses the formation and breakage of disulfide bonds between cysteine residues to produce a stable conformation.

There are two possible mechanisms for the insertion of integral membrane proteins having a single transmembrane segment.
1) Type I: Insertion of a polypeptide with both a terminal ER signal sequence and an internal stop-transfer sequence.
The terminal peptide is eventually cut off, leaving a transmembrane protein with its N-terminus in the ER lumen and its C-terminus in the cytosol.
2) Type II:  Insertion of a polypeptide with only a single, internal start transfer sequence, which both starts polypeptide transfer and anchors itself permanently in the membrane.
The amino-carboxyl orientation of the completed protein depends on the orientation of the start-transfer sequence when it first inserts into the translocation apparatus.

Posttranslational import allows some polypeptides to enter organelles after protein synthesis.
Like cotranslational import into the ER, posttranslational import into a mitochondrion (and chloroplast) involves a signal sequence (called a transit sequence), a membrane receptor, pore-forming membrane proteins, and a peptidase.
Polypeptides being imported into the mitochondrion span both membranes at the same time.
This was demonstrated in a cell-free import system incubated on ice in which the polypeptides begin to penetrate the mitochondrion but then stall.
The transit sequence is cleaved by the transit peptidase present in the matrix, indicating that the N-terminus of the polypeptide is within the mitochondrion.
At the same time, most of the polypeptide molecule is can be attacked by exogenously added proteolytic enzymes on the outside of the mitochondrion.
Therefore, the polypeptide must span both membranes transiently during import at a contact site between the two membranes.

However, in the mitochondrion, the membrane receptor recognizes the signal sequence directly without the intervention of a cytosolic SRP.
Furthermore, chaperone proteins play several crucial roles in the mitochondrial process:
1) Chaperones keep the polypeptide partially unfolded after synthesis in the cytosol so that binding of the transit sequence and translocation can occur.
2) Chaperones drive the translocation itself by binding to and releasing from the polypeptide within the matrix, an ATP-requiring process and
3) Chaperones often help the polypeptide fold into its final conformation.

Polypeptides synthesized on cytosolic ribosomes but destined for either the intermembrane space or the inner membrane of the mitochondrion require two separate targeting sequences (both located at the N-terminus).
1) The polypeptide is directed to a contact (translocation) site on the mitochondrion by a positively charged or amphipathic transit sequence.
2) Cleavage of the transit sequence by a peptidase in the mitochondrial matrix uncovers a highly hydrophobic second signal sequence.
3) This second signal sequence causes the polypeptide to be inserted into the inner membrane in the same way that mitochondrially encoded polypeptides are targeted to this membrane.
4) The remainder of the polypeptide is then moved across the membrane into the intermembrane space (or into the inner membrane for integral inner membrane proteins).
5) Cleavage by a second peptidase can release the polypeptide into the intermembrane space leaving the signal sequence behind in the inner membrane.

Notes prepared from Becker's World of the Cell, 9th edition
Hardin & Bertoni, 2015
Figures copyright of Pearson Education Inc.
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