translation
Translation of mRNA is the biological polymerization of amino acids into polypeptide chains.
This process occurs only in association with ribosomes.
tRNA is the molecule that adapts/binds specific triplet codons in mRNA to their correct amino acids.
tRNA contains within its nucleotide sequence 3 consecutive ribonuceotides complementary to the codon, called anticodon.
Ribosomal structure
There are about 10,000 ribosomes in bacteria and many more in eukaryotes.
Ribosomes consist of two subunits, one large and one small.
Both subunits consist of one or more molecules of rRNA and some ribosomal proteins.
When the two subunits associate with each other to form a single ribosome, it is sometimes called a monosome.
In prokaryotes the monosome is 70S particle (50S and 30S), in eukaryotes it is 80S (60S and 40S).
The genes responsible for the production of rRNA (rDNA) are part of the moderately repetitive DNA and are clustered near the ends of chromosomes 13, 14, 15, 21 and 22.
tRNA structure
It is composed of only 75 to 90 nucleotides.
tRNAs have almost identical structure both in bacteria and in eukaryotes.
tRNAs are transcribed as larger precursors which are later cleaved into mature tRNA molecules.
tRNA has number of unique nucleotides.
These unique nucleotides are formed by the modification of bases (G, C, A and U) present in RNA after transcription.
These include: inosinic acid, ribothymidylic acid and pseudourudune among others…
Robert Holley proposed a two-dimentional cloverleaf model of tRNA.
He arranged the linear model in such a way that several stretches of base-pairing would result.
The arrangement created several paired stems and unpaired loops resembling the shape of the cloverleaf.
Loops contained modified bases that did not form base pairs.
Cloverleaf model of tRNA
Triplet GCU, GCC and GCA specify alanine.
Holley found an anticodon sequence complementary to one of these codons in his tRNAala molecule.
He found it in the form of CGI (3’ to 5’ direction) in one loop of the cloverleaf.
Nitrogenous base I (inosinic acid) can form hydrogen bonds with U, C or A, the third members of the alanine triplets.
Thus, anticodon loop was established!
Anticodon-codon base-pairing rules
tRNA also contains unusual base such as inosine (I) that can pair with A, U or C.
Thus, GCI anticodon of tRNA can recognize GCU, GCC and GCA which code for alanine.
At 3’ end, all tRNAs contain the sequence CCA, which is the site of amino acid binding during translation. All tRNAs contain G at the 5’ end of the molecule.
Each tRNA contains an anticodon complementary to the known amino acid codon for which it is specific.
All anticodon loops are present in the same position of the cloverleaf!
Charging tRNA
Before translation can proceed, the tRNA molecules must be chemically linked to their respective amino acids.
This activation process, called charging or aminoacylation, occurs under the direction of enzymes called aminoacyl tRNA synthetases.
Because of the ability of the third member of a triplet code to “wobble,” the minimum number of different tRNAs required is only 31.
“the wobble” allows the anticodon of a single tRNA to pair with more than one mRNA codons
In the initial step, the amino acid is converted to an activated form, reacting with ATP to create an aminoacyladenylic acid.
During the next step, the amino acid is transferred to the appropriate tRNA and bonded covalently to the adenine residue at the 3’ end.
The charged tRNA may then participate directly in protein synthesis.
Aminoacyl tRNAsynthetases are highly specific enzymes because they recognize only one amino acid and only the tRNAscorresponding to that amino acid, calledisoacceptingtRNAs.
Translation is divided into 3 steps: 1. Initiation; 2. Elongation; 3. Termination
1. Translation initiation is a process in which mRNA, tRNA and small and large ribosomal subunits associate with each other to form a complex.
This process is facilitated by a Shine-Delgarnosequence in the mRNA, which is complementary to a component of the small ribosomal subunit called 16S ribosomal RNA.
Though a shorter sequence is shown here, the Shine-Delgarno sequence is actually 9 nucleotides long.
Initiation factor 3 (IF3) also facilitates the binding of the mRNA to the small ribosomal subunit.
Afterwards initiation factor 2 (IF2) binds to the complex, and promotes the binding of the tRNA for N-formyul methionine to the complex.
Translation initiation is completed when the large ribosomal subunit binds and IF2 and IF3 are released.
Later, when translation is completed, IF1 is needed for the dissociation of the complex.
2. Translation elongationbegins with the binding of a tRNA, which recognizes the next codon in the mRNA, to the A site of the ribosome. (A site - acceptor site, P site - peptidyl site, E site - exit site)
Once the tRNA binds in the A site of the ribosome, the polypeptide chain is moved from the tRNA in the P site to the amino acid attached to the tRNA in the A site.
Peptidyl transferase, a protein/RNA complex present in the 50S ribosomal subunit, catalyzes the formation of this new peptide bond between the amino acids.
The ribosome than is translocated to the next codon.
This places the empty tRNA molecule in the E site of the ribosome, and moves the tRNA containing the growing polypeptide chain in the P site. The next codon in the mRNA chain is positioned in the A site.
The uncharged or empty tRNA in the E site then leaves the ribosome and a cycle of chain elongation is completed.
Through subsequent cycles of chain elongation the polypeptide chain continues to elongate one amino acid at a time.
3. Termination begins when a stop codon appears in the A-site.
Since there is no tRNA corresponding to the stop codon, a release factor binds in the A-site.
The binding of the release factor causes the polypeptide chain to be cleaved from the tRNA, completing the synthesis of the polypeptide.
The polypeptide is released.
Then the tRNA is released.
In the final step, the two ribosomal subunits and the mRNA dissociate from each other.
The termination process is completed.
If a termination codon should appear in the middle of an mRNA molecule as a result of mutation, the same process occurs, and the polypeptide chain is prematurely terminated (nonsense mutation).
Translation in eukaryotes is more complex
One main difference between translation in prokaryotes and eukaryotes is that in eukaryotes, translation occurs on larger ribosomes whose rRNA and protein components are more complex than those of prokaryotes.
Eukaryotic mRNAs are much longer-lived than their prokaryotic counterparts.
Most exist for hours before degradation by nucleases in the cell.
Transcription and translation happen simultaneously in prokaryotes, but in eukaryotes these two processes are separated both specially and temporally.
In eukaryotes transcription occurs in the nucleus and translation in the cytoplasm.
In eukaryotes the 5’ end of the mRNA is capped with a 7-methylguanosine (7mG) residue (it is absent in prokaryotes).
The presence of 7mG cap is essential for efficient translation, because RNAs that lack the cap are translated poorly.
Proteins
Alkaptonuria
Metabolic disorder
Patients cannot metabolize the alkapton 2,5-dihydroxyphenilacetic acis (homogenistic acid).
It accumulates in the cells and tissues and is excreted in the urine.
The molecule has a black color and is easily detectable in the diapers of the newborn babies.
Homogenistic acid accumulates in the cartilages, causing ears and nose to be black.
The disease is not serious, but it persists throughout life.
In 1902 Sir Archibald Garrod, who studies this disease, analyzed 32 cases and found that 19 cases existed in 7 families.
In several instances the parents were not affected, but were first cousins.
Garrod concluded that this disease was a result of impaired metabolism and that hereditary material controls chemical reactions in the body.
However, for almost 30 years from his publications, geneticists didn’t see the relationship between the genes and the enzymes.
Phenilketonuria - PKU
Autosomal-recessive metabolic disorder that if not treated results in mental retardation.
Patients are unable to convert amino acid phenylalanine to tyrosine.
These molecules differ from each other only by a single hydroxyl (OH) group that is present in tyrosine but absent in phenylalanine.
This reaction is catalized by an enzyme phenylalanine hydroxylase which is inactivated in affected individuals.
Phenylalanine hydroxylase is active at 30% level in heterozygous carriers.
The enzyme is expressed in the liver, but the brain is damaged.
Normal blood level of phenilalanine in blood is 1mg/100mL, but in PKU patients it can be 50mg/mL.
Phenylalanine acumulates and is converted to phenylpiyuvic acid.
Both phenylalanine and phenylpiyuvic acid enter cerebrospinal fluid and damage the brain, causing mental retardation.
Newborn screening is done in many countries
Dietary restriction should be made before 4 weeks after the birth is the screening result is positive
Hundreds of metabolic diseases are known today resulting from mutant genes!!!
1940s – one gene:one enzyme hypothesis – not accepted… (Nearly all enzymes are proteins, but not all proteins are enzymes. All proteins are specified by the information stored in genes)
One gene:one protein hypothesis (Proteins aften show a substructure consisting of two or more polypeptide chains; each polypeptide chain is encoded by a separate gene)
One gene:one polypeptide hypothesis
Sickle-cell anemia
The first single-gene disorder to be strudied at molecular level!!!
RBCs of affected individuals become elongated and curved (sickle) because of the polymerization of hemoglobin instead of being biconcave and flexible.
The Hb molecule contains four subunits: two a or a-like chains and two β or β-like chains.
Patients suffer attacks when RBCs aggregate in venous capillaries, where oxygen tension is low.
Different tissues are damages depending on where the damage happened.
The attacks or crisis are very painfulю. Disease may cause death if untreated
Mutation is caused by a single amino acid substitution that changes glutamic acid to valine at the 6th position in the beta chain of the Hb molecule.
Protein structure
Structure of proteins provides the basis of complexity and diversity of their cellular activities.
What’s the difference between polypeptides and proteins???
Both are molecules composed of amino acids.
BUT…
Polypeptides are assambled on the ribosomes during translation and they are the precursors of proteins.
After translation polypeptides fold up and takes a three-dimensional shape. Sometimes several polypeptides interact to produce its final conformation and as the molecule becomes fully functional, it is called a protein.
The polypeptide chains of proteins are linear non-branched polymers.
There are 20 amino acids that serve as subunits of proteins.
Each amino acid consists of:
1. carboxyl group;
2. amino group;
3. R (radical) group - The R group gives each amino acid it chemical identity
Amino acids are bound covalently to a central carbon (C) atom.
Amino acids are divided into 4 major classes:
Nonpolar: Hydrophobic
Polar: Hydrophilic
Polar: Positively charged
Polar: Negatively charged
Water gets attracted to the polar (hydrophilic) amino acids
Nonpolar (hydrophobic) amino acids hide from the water
Positively and negatively charged amino acids attract to each other
Interaction of different types of amino acids define the final structure/shape/ conformation of the protein!!!
Because polypeptides are long polymers and because each position may be occupied by any 1 of the 20 amino acids, enormous variation in chemical conformation and activity are possible!!!
But proteins can function properly only if their structure is proper too!!!
Amino group of one amino acid reacts with carboxyl group of another amino acid during dehydration reaction, releasing water molecule.
Two amino acids linked together is called a dipeptide.
Three amino acids- tripeptide.
10 or more amino acids linked together by peptide bond – polypeptide.
No matter how long a polypeptide is, it will contain a free amino group at one end and a free carboxyl group at the other end.
The sequence of amino acids in the linear sequence of the polypeptide constitutes a primary structure.
Secondary structure is a highly regular repetitive configuration in space and assumes that amino acids lye close to each other in the polypeptide chain. alpha helix is spiral and is stabilized by multiple hydrogen bonds. In beta sheet structure a polypeptide folds back on itself or several chains run parallel or anti-parallel fashion next to each other. Hydrogen bonds form between adjacent chains.
Tertiary structure defines the tree-dimentional conformation of the entire chain in space. Each protein twists and turns and loops around itself in a very particular fashion, characteristic to the specific protein.
Quaternary structure refers to a protein that is composed of more than one polypeptide chain and indicates position of various chains in relation to one another.
This type of protein is oligomeric and each chain is a subunit.
Protein folding and misfolding
It was believed that protein folding was a random process where a linear molecule exiting the ribosome achieved a three-dimentional stable conformation based only on its amino acid composition.
But in some proteins correct folding depends on other proteins, called chaperons.
Chaperons mediate the folding process by preventing formation of incorrect configurations.
They can bind to the protein, BUT they do not become the part of protein’s final product of like enzymes!
Chaperons are discovered in all organisms.
They are even present in mitochondria and chloroplasts.
But sometimes, even in the presence of chaperons, protein misfolding may still occur.
As misfolded proteins are transported out from the endoplasmic reticulum to the cytoplasm, they are “caught” by another class of proteins called ubiquitins and the misfolded protein is degraded.
Protein folding is a critically important process in the organism!
- Misfolded protein may be nonfunctional
- Misfolded proteins can accumulate in the body and damage the cells and the tissues.
Ex: Scrapie in sheep and goat, mad-cow disease, Creutzfeldt-Jakob disease in humans – caused by prions, which are aggregates of a misfolded protein and cause death!!!
Usually the normal protein (PrPc) has alpha-helix conformation, but the misfolded protein (PrPsc) folds into beta-pleated sheet.
The protein is synthesizedin the neurons!
When the abnormal molecule (PrPsc) contacts the normal molecule (PrPc) , the normal protein turns into the abnormal conformation.
This process continues as a chain reaction and destroies the brain! ☹
Ex: Sickle-cell anemia, where beta chain of the polypeptide is altered because of one amino acid change and causes the molecule to aggregate within erythrocyte.
Huntington disease, Alzhimer’s diseas and Parkinson disease - lead to the formation of abnormal protein aggregate in the brain.
Proteins function in may different ways
Proteins are the most abundant macromolecules found in cells.
They are the end products of genes and play many diverse roles in the body!
Hemoglobin and myoglobin (respiratory pigments) transport oxygen
Collagen and keratin are structural proteins associated with skin, connective tissue and hair
Actin and myosin are contractile proteins found in muscle tissue
Immunoglobulins function in the immune system of vertebrates
Transport proteins are involved in the movement of molecules across emmbranes
Hormones regulate various types of chemical activity
The largest group of proteins with related functions are enzymes!
Enzymes catalyze chemical reactions within living cells.
They increase the rate at which chemical reaction reaches equilibrium, BUT do not change the end-point of the chemical equilibrium!
Usually molecules must increase kinetic energy to interact with each other and this state is achieved with very high temperatures.
But enzymes allow biological reactions to occur at lower physiological temperatures.
Enzymes make life possible!!!
Enzymes have so called active site, with which they can bind to reactants or substrates and promote interaction.
Enzymatically catalyzed reactions control metabolic activities in the cell.
Catabolism – is the degradation of large molecules into smaller, simpler ones with the release of chemical energy.
Anabolism – is the set of metabolic pathways that construct nucleic acids, proteins, lipids and carbohydrates from smaller units. It requires energy.