TRANSLATION

BASALINAGAPPA DMLT,.BMLT,.BSBT.,

M.SC. MEDICAL BIOCHEMISTRYDEPT. OF BIOCHEMISTRY J S S MEDICAL COLLEGE

Translation,

1. mRNA : RBS, start-stop codon , 2. Ribosome : rRNA + protein + protein 3. tRNA : tRNA charging charging4. Amino acid : genetic code

• Translation, the second part of the central dogma of molecular biology, describes how the genetic code is used to make amino acid chains.

• In this lesson, explore the mechanics involved in polypeptide synthesis.

• Learn the three major steps of translation as you watch tRNA, mRNA, and ribosomes go to work.

• Translation is the process that takes the information passed from DNA as messenger RNA and turns it into a series of amino acids bound together with peptide bonds.

• It is essentially a translation from one code (nucleotide sequence) to another code (amino acid sequence).

• The ribosome is the site of this action, just as RNA polymerase was the site of mRNA synthesis.

• The ribosome matches the base sequence on the mRNA in sets of three bases (called codons) to tRNA molecules that have the three complementary bases in their anticodon regions.

• Again, the base-pairing rule is important in this recognition (A binds to U and C binds to G). The ribosome moves along the mRNA, matching 3 base pairs at a time and adding the amino acids to the polypeptide chain.

• When the ribosome reaches one of the "stop" codes, the ribosome releases both the polypeptide and the mRNA. This polypeptide will twist into its native conformation and begin to act as a protein in the cells metabolism.

• To understand basic steps in translation are:• The ribosome binds to mRNA at a specific area.• The ribosome starts matching tRNA anticodon sequences to

the mRNA codon sequence.• Each time a new tRNA comes into the ribosome, the amino

acid that it was carrying gets added to the elongating polypeptide chain.

• The ribosome continues until it hits a stop sequence, then it releases the polypeptide and the mRNA.

• The polypeptide forms into its native shape and starts acting as a functional protein in the cell.

Types of RNA:

• mRNA - messenger RNA is a copy of a gene. It acts as a photocopy of a gene by having a sequence complementary to one strand of the DNA and identical to the other strand. The mRNA acts as a busboy to carry the information stored in the DNA in the nucleus to the cytoplasm where the ribosomes can make it into protein.

• tRNA - transfer RNA is a small RNA that has a very specific secondary and tertiary structure such that it can bind an amino acid at one end, and mRNA at the other end. It acts as an adaptor to carry the amino acid elements of a protein to the appropriate place as coded for by the mRNA.

• rRNA - ribosomal RNA is one of the structural components of the ribosome. Its sequence is the compliment of regions in the mRNA so that the ribosome can match with and bind to an mRNA it will make a protein from.

The Three Steps of Translation

• Translation is the second step in the central dogma that describes how the genetic code is converted into amino acids.

• We've talked about how the mRNA codes are recognized by tRNA and how the amino acids are linked together by peptide bonds.

• A chain of amino acids is also called a polypeptide. Polypeptides are assembled inside the ribosomes, which are tiny organelles on the rough ER of a cell.

• Now that we're learning more about the mechanics of translation, we're going to have to start putting the pieces together.

• We already understand the role of the ribosome and the amino acids in the process of translation, but how does polypeptide assembly actually occur? .

There are three important steps to the process of translation

• There's a • Beginning step, initiation, • a middle step, called elongation,• a final step, called termination.

• These three words may sound familiar to you. The same terms are used in transcription to describe the steps involved in making the mRNA strand.

• But, here in translation, we're making a polypeptide strand. • In either case, we're making a long molecule out of a chain

of smaller subunits. • So, whether we're referring to transcription or translation,

the three terms accurately describe the mechanics of the process.

• Let's walk through each step, one at a time

Initiation

• We'll start with initiation. During initiation, the mRNA, the tRNA, and the first amino acid all come together within the ribosome.

• The mRNA strand remains continuous, but the true initiation point is the start codon, AUG. Remember that the start codon is the set of three nucleotides that begins the coded sequence of a gene.

• Remember also that the start codon specifies the amino acid methionine. So, methionine is the name of the amino acid that is brought into the ribosome first.

And, how did methionine get itself to the ribosome? By attaching to the tRNA that contains the right anticodon. The anticodon for AUG is UAC. We know that because of the rules of complementary base pairing. The tRNA with the anticodon UAC will automatically match to the codon AUG, bringing the methionine along for the ride. So, there you have it - mRNA is attached to tRNA, and tRNA is attached to methionine. That's initiation.

Elongation

• The next step makes up the bulk of translation. It's called elongation, and it's the addition of amino acids by the formation of peptide bonds.

• Elongation is just what it sounds like: a chain of amino acids grows longer and longer as more amino acids are added on. This will eventually create the polypeptide.

• Now that we've begun with the start codon, the mRNA shifts a little through the ribosome so that the next codon is up for grabs.

• Let's say the next codon is UAU. So, now we need a tRNA that has the matching anticodon, AUA. Oh, look! Here's a tRNA with the right anticodon, and it's brought along a tyrosine.

• Tyrosine is the amino acid that is specified by the codon UAU. • The tRNA attaches to the mRNA in the ribosome and lines up

tyrosine right next to the waiting methionine. A peptide bond forms between the two amino acids.

• Then, the first tRNA leaves everyone else behind and floats off to find more work to do. Poor methionine! Now it's just drifting around like a lonely kite in the wind! That tRNA left methionine hanging by only one anchor: its peptide bond with tyrosine.

• The tyrosine is still attached to its own tRNA, which, in turn, is clinging to the mRNA inside the ribosome.

• Already we can see the beginnings of a polypeptide elongating outward.

• Should we walk through that process one more time? Let's keep everything just as we have it here and move on to add our third amino acid. mRNA shifts over again, and now the third codon is ready for a match.

• What's that codon? CAC. Here comes a tRNA with the matching anticodon, GUG.

• It's also brought us a histidine, since CAC codes for histidine.

• The tRNA's anticodon matches up with the mRNA's codon, putting the histidine in perfect position for making a peptide bond with tyrosine.

The ribosome then moves one triplet forward and a new tRNA+amino acid can enter the ribosome and the procedure is repeated.

How is the right tRNA Chosen• Non-matching tRNA's may also enter the A site. • Why doesn't this cause wrong amino acids to be added to the

chain?• Well, non-matching tRNA's may be able to enter the A site...but

in general, they don't get to stay there. In a careful process that never fails to fascinate me, each tRNA is escorted by helper proteins, and only a tRNA that's a perfect match for the codon will be "released" into the A slot by its choosy helpers.^7 7 start superscript, 7, end superscript

• A molecule of the energy storage molecule guanosine triphosphate (GTP) is used up to release the tRNA, which is why the diagram shows this step as needing GT

What are the N and C terminus

• Great question! Amino acids have an amino group {−NH 2 } at one end and of a carboxyl group {-COOH} at the other:

• A polypeptide is made up of amino acids connected in a chain.

• Although the amino and carboxyl groups of most amino acids in the chain will be tied up in peptide bonds, the amino acids at the very ends of the chain will each have a free chemical group.

• One end of the polypeptide has an exposed amino group. This end is called the N-terminus ,for the nitrogen atom of the amino group.

• The other end of the polypeptide has an exposed carboxyl group. This end is called the C-terminus, for the carbon atom of the carboxyl group.

• The first amino acid in a polypeptide (the methionine carried by the first tRNA) lies at the N-terminus, and new amino acids are progressively added at the C-terminus:

• But...odds are we may want a longer polypeptide than two amino acids. How does the chain continue to grow? Once the peptide bond is formed, the mRNA is pulled onward through the ribosome by exactly one codon. This shift allows the first, empty tRNA to drift out via the E ("exit") site. It also exposes a new codon in the A site, so the whole cycle can repeat.

Termination

• Translation ends when one of three stop codons, UAA, UAG, or UGA, enters the A site of the ribosome.

• There are no aminoacyl tRNA molecules that recognize these sequences.

• Instead, release factors bind to the P site, catalyzing the release of the completed polypeptide chain and separating the ribosome into its original small and large subunits

• To meet this requirement they make many mRNA copies of the corresponding gene and have many ribosomes working on each mRNA. After translation the protein will usually undergo some further modifications before it becomes fully active.

At a given time, more than one ribosome may be translating a single mRNA molecule. The resulting clusters of ribosomes, which resemble beads on a string, are called polysomes

• When the ribosome reaches one of three stop codons, for example UGA, there are no corresponding tRNAs to that sequence.

• Instead termination proteins bind to the ribosome and stimulate the release of the polypeptide chain (the protein), and the ribosome dissociates from the mRNA.

• When the ribosome is released from the mRNA, its large and small subunit dissociate. The small subunit can now be loaded with a new tRNA+methionine and start translation once again.

• Some cells need large quantities of a particular protein.

Easy To Remember Translation Steps

Step 1 of Translation : mRNA attaches to the ribosome.Step 2 of Translation : tRNA's attach to free amino acids in the cytoplasmic "pool" of amino acids.step 3of Translation t: RNA carries its specific amino acid to the ribosome.

Step 4 of Translation : tRNA "delivers" its amino acid based on complementary pairing of a triplet code (anticodon) with the triplet code (codon) of the mRNA.

Step 5 of Translation : Enzyme "hooks" the amino acid to the last one in the chain forming a peptide bond.

Step 6 of Translation : Protein chain continues to grow as each tRNA brings in its amino acid and adds it to the chain.

Elongation & Termination of protein biosynthesis

Eukaryotic Translation• Eukaryotic translation is very similar overall to prokaryotic

translation. There are a few notable differences, These include the followings:

•Eukaryotic mRNAs do not contain a Shine-Delgarno sequence. Instead, ribosomal subunits recognize and bind to the 5' cap of eukaryotic mRNAs. In other words, the 5' cap takes the place of the Shine-Delgarno sequence.

• Eukaryotes do not use formyl methionine as the first amino acid in every polypeptide; ordinary methionine is used.

• Eukaryotes do have a specific initiator tRNA, however. • Eukaryotic translation involves many more protein

factors than prokaryotic translation (For example, eukaryotic initiation involves at least 10 factors, instead of the 3 in prokaryotes.)

• Choramphenicol inhibits prokaryotic peptidyl Transferase

• Clindamycin and Erythromycin bind irreversibly to a site on the 50 s subunit of the bacterial ribosome thus inhibit translocation.

• Diphtheria toxin inactivates the eukaryotic elongation factors thus prevent translocation

Inhibitors of protein synthesis

• The tetracyclines (tetracycline, doxycycline, demeclocycline, minocycline, etc.) block bacterial translation by binding reversibly to the 30S subunit and distorting it in such a way that the anticodons of the charged tRNAs cannot align properly with the codons of the mRNA.

• Puromycin structurally binds to the amino acyl • t RNA and becomes incorporated into the growing

peptide chain thus causing inhibition of the further elongation.

Translation: Summary of Key Points Translation is the synthesis of a polypeptide using the

information encoded in an mRNA molecule. The process involves mRNA, tRNA, and ribosomes.

tRNA has a unique structure that exposes an anticodon, which binds to codons in an mRNA, and an opposite end that binds to a specific amino acid.

Binding of an amino acid to a tRNA is carried out by an enzyme called amino acyl tRNA synthetase in a process called charging.

Translation consists of three basic steps: initiation, elongation, and termination.

Initiation involves the formation of the ribosome/mRNA/initiator tRNA complex

Elongation is the actual synthesis of the polypeptide chain, by formation of peptide bonds between amino acids.

Termination dissociates the translation complex and releases the finished polypeptide chain. Each of these steps requires the activity of a specific set of protein factors in addition to the ribosome, tRNA, and mRNA.

Post Translational ModificationsTrimming removes excess amino acids.Phosphorylation may activate or inactivate the proteinGlycosylation targets a protein to become a part of the

plasma membrane , or lysosomes or be secreted out of the cell

Hydroxylation such as seen in collagen is required for acquiring the three dimensional structure and for imparting strength

Defective proteins or destined for turn over are marked for destruction by attachment of a Ubiquitin protein. Proteins marked in this way are degraded by a cellular component known as the Proteasome.

• Once the protein has been synthesized by the ribosome from its corresponding mRNA in the cytosol, many proteins get directed towards the endoplasmic reticulum for further modification.

• Certain N and C terminal sequences are often cleaved in the ER after which they are modified by various enzymes at specific amino acid residues. These modified proteins then undergo proper folding to give the functional protein.

• Most of the proteins that are translated from mRNA undergo chemical modifications before becoming functional in different body cells. The modifications collectively, are known as post-translational modifications.

• The protein post translational modifications play a crucial role in generating the heterogeneity in proteins and also help in utilizing identical proteins for different cellular functions in different cell types.

• How a particular protein sequence will act in most of the eukaryotic organisms is regulated by these post translational modifications.

• Post translational modifications occurring at the peptide terminus of the amino acid chain play an important role in translocating them across biological membranes.

• These include secretory proteins in prokaryotes and eukaryotes and also proteins that are intended to be incorporated in various cellular and organelle membranes such as

• lysosomes, • chloroplast, • mitochondria and plasma membranes.

• Expression of proteins is important in diseased conditions. Post translational modifications play an important part in modifying the end product of expression and contribute towards biological processes and diseased conditions.

• The amino terminal sequences are removed by proteolytic cleavage when the proteins cross the membranes.

• These amino terminal sequences target the proteins for transporting them to their actual point of action in the cell.

Why PTM is necessary

• Stability of protein.• Biological activity.• Protein targeting.• Protein signaling.

• As noted above, the large number of different PTMs precludes a thorough review of all possible protein modifications.

• Therefore, this overview only touches on a small number of the most common types of PTMs studied in protein research today.

• Furthermore, greater focus is placed on phosphorylation, glycosylation and ubiquitination, and therefore these PTMs are described in greater detail on pages dedicated to the respective PTM

Importance of PTMs

• Play a crucial role in generating the heterogeneity in proteins.

• Help in utilizing identical proteins for different cellular functions in different cell types.

• Regulation of particular protein sequence behavior in most of the eukaryotic organisms.

• Play an important part in modifying the end product of expression.

• Contribute towards biological processes and diseased conditions.

• Translocation of proteins across biological membranes

• There are several types of post translational modifications that can take place at different amino acid residues.

• The most commonly observed PTMs include phosphorylation,

• glycosylation, • methylation as well as• hydroxylation and acylation. • Many of these modifications, particularly

phosphorylation, serve as regulatory mechanisms for protein action.

Phosphorylation• Reversible protein phosphorylation, principally on serine,

threonine or tyrosine residues, is one of the most important and well-studied post-translational modifications.

• Phosphorylation plays critical roles in the regulation of many cellular processes including cell cycle, growth, apoptosis and signal transduction pathways.

• Phosphorylation is the most common mechanism of regulating protein function and transmitting signals throughout the cell. While phosphorylation has been observed in bacterial proteins, it is considerably more pervasive in eukaryotic cells.

• It is estimated that one-third of the proteins in the human proteome are substrates for phosphorylation at some point (1). Indeed, phosphoproteomics has been established as a branch of proteomics that focuses solely on the identification and characterization of phosphorylated proteins.

Addition of phosphate group to a protein.Principally on serine, threonine or tyrosine residues.Also known as Phospho regulation.Critical role in cell cycle, growth, apoptosis and signal transduction pathways.

• Phosphorylation of amino acid residues is carried out by a class of enzymes known as kinases that most commonly modify side chains of amino acids containing a hydroxyl group.

• Phosphorylation requires the presence of a phosphate donor molecule such as ATP, GTP or other phoshorylated substrates.

• Serine is the most commonly phosphorylated residue followed by threonine and tyrosine.

• Removal of phosphate groups is carried out by the phosphatase enzyme and thus this forms one of the most important mechanisms for regulation of proteins.

Glycosylation• Glycosylation involves the enzymatic addition of

saccharide molecules to amino acid side chains. This can be of two types – N-linked glycosylation, which links sugar residues to the amide group of aspargine and Olinked glycosylation, which links the sugar moieties to the hydroxyl groups of serine or threonine.

• Suitable glycosyl transferase enzymes catalyze these reactions.

• Sugar residues that are attached most commonly include galactose, mannose, glucose, Nacetylglucosamine, Nacetylgalactosamie as well as fucose

The covalent attachment of oligosaccharidesAddition of glycosyl group or carbohydrate group to a protein.Principally on Asparagine, hydroxylysine, serine or threonine.Significant effect on protein folding, conformation, distribution, stability and activity

Hydroxylation• During the formation of collagen, the amino

acids proline & lysine are respectively converted to hydroxyproline & hydroxylysine.

• This hydroxylation occurs in the endoplasmic reticulum & requires vitaminC.

Ubiquitination

• Ubiquitin is a small regulatory protein that can be attached to the proteins and label them for destruction.

• Effects in cell cycle regulation, control of proliferation and differentiation, programmed cell death (apoptosis), DNA repair, immune and inflammatory processes and organelle biogenesis.

Ubiquitin cycle

S-Nitrosylation

• Nitrosyl (NO) group is added to the protein.• NO a chemical messanger that reacts with free

cysteine residues to form S-nitrothiols.• Used by cells to stabilize proteins, regulate

gene expression.

Alkylation/Methylation

• Addition of methyl group to a protein.• Usually at lysine or arginine residues.• Binds on nitrogen and oxygen of proteins• Methyl donor is S-adenosylmethionine (SAM)• Enzyme for this is methyltransferase• Methylation of lysine residues in histones in

DNA is important regulator of chromatin structure

SAM (S-adenosyl methionine) is converted into SAH(S-adenosyl homocysteine)

N-Acetylation

• Addition of acetyl group to the nitrogen.• Histones are acetylated on lysine residues in the

N-terminal tail as a part of gene regulation.• Involved in regulation of transcription factors,

effector proteins, molecular chaperons and cytoskeletal proteins.

• Methionine aminopeptidase (MAP) is an enzyme responsible for N-terminal acetylation

Lipidation

• Lipidation attachment of a lipid group, such as a fatty acid, covalently to a protein.

• In general, lipidation helps in cellular localization and targeting signals, membrane tethering and as mediator of protein-protein interactions

Identification of PTM modifications

• Mass spectrometry• HPLC analysis • Incorporation of radioactive groups by addition to

growing cells– e.g., 75Se-labeling and chromatographic isolation

of proteins• Antibody cross-reactivity– e.g., antibody against phosphotyrosine

• Polyacrylamide gel electrophoresis (PAGE