Genetics Block 2 - PROTEIN SYNTHESIS (TRANSLATION) in progress

Components required for the synthesis of a protein

(I). amino acids found in the finished product (II). mRNA to be translated (III). tRNA (IV). Functional ribosomes (V). Energy sources (VI). Enzymes (VII). Protein factors for initiation, elongation, & termination of polypeptide chain.

Initiation codon

AUG is recognized by a special initiator tRNA. Recognition is facilitated by IF-2 (bound to GTP) in prokaryotes and eIF2-GTP (plus additional eIF) in eukaryotes. The amino acid-charged initiator tRNA enters the ribosomal P site, and GTP is hydrolyzed to GDP. The initiator tRNA is the only tRNA recognized by eIF-2, and the only tRNA to go directly to the P site. In bacteria and in mitochondria, the initiator tRNA carries an N-formylated methionine. The formyl group is added to the methionine after that amino acid is attached to the initiator tRNA by the enzyme transformylase, which uses N10-formyl tetrahydrofolate as the carbon donor. In eukaryotes, the initiator tRNA carries a methionine that is not formylated. In both prokaryotic cells and mitochondria, this N-terminal methionine is usually removed before translation is completed.

tRNA structure

Amino acid attachment site: Each tRNA molecule has an attachment site for a specific (cognate) amino acid at its 3′-end. The carboxyl group of the amino acid is in an ester linkage with the 3′-hydroxyl of the ribose moiety of the adenosine nucleotide in the —CCA sequence at the 3′-end of the tRNA. [Note: When a tRNA has a covalently attached amino acid, it is said to be charged; when tRNA is not bound to an amino acid, it is described as being uncharged.] The amino acid that is attached to the tRNA molecule is said to be activated.

transfer RNA

At least one specific type of tRNA is required per amino acid. In humans, there are at least fifty species of tRNA, whereas bacteria contain thirty to forty species. Because there are only twenty different amino acids commonly carried by tRNA, some amino acids have more than one specific tRNA molecule. This is particularly true of those amino acids that are coded for by several codons.

CODON RECOGNITION BY tRNA

Correct pairing of the codon in the mRNA with the anticodon of the tRNA is essential for accurate translation. Some tRNAs recognize more than one codon for a given amino acid. Antiparallel binding between codon and anticodon Binding of the tRNA anticodon to the mRNA codon follows the rules of complementary and antiparallel binding, that is, the mRNA codon is "read" 5′→3′ by an anticodon pairing in the "flipped" (3′→5′) orientation. [Note: When writing the sequences of both codons and anticodons, the nucleotide sequence must ALWAYS be listed in the 5′→3′ order.]

A site

During translation, the A site binds an incoming aminoacyl-tRNA as directed by the codon currently occupying this site. This codon specifies the next amino acid to be added to the growing peptide chain.

Anticodon

Each tRNA molecule also contains a three-base nucleotide sequence that recognizes a specific codon on the mRNA. This codon specifies the insertion into the growing peptide chain of the amino acid carried by that tRNA.

degeneracy

The genetic code is degenerate (sometimes called redundant). Although each codon corresponds to a single amino acid, a given amino acid may have more than one triplet coding for it. ex: arginine is specified by six different codons

Amino-acyl Trna Synthetases

This family of enzymes is required for attachment of amino acids to their corresponding tRNA. Each member of this family recognizes a specific amino acid and the tRNA that correspond to that amino acid (isoaccepting tRNA). These enzymes thus implement the genetic code because they act as molecular dictionaries that can read both the three-letter code of nucleic acids and the twenty-letter code of amino acids. Each aminoacyl-tRNA synthetase catalyzes a two-step reaction that results in the covalent attachment of the carboxyl group of an amino acid to the 3′-end of its corresponding tRNA. The overall reaction requires adenosine triphosphate (ATP), which is cleaved to adenosine monophosphate (AMP) and inorganic pyrophosphate (PPi). The extreme specificity of the synthetase in recognizing both the amino acid and its specific tRNA contributes to the high fidelity of translation of the genetic message. In addition, the synthetases have a "proofreading" or "editing" activity that can remove mischarged amino acids from the enzyme or the tRNA molecule.

genetic code analogy

genetic code is a dictionary that identifies the correspondence between a sequence of nucleotide bases and a sequence of amino acids. Each individual word in the code is composed of three nucleotide bases. These genetic words are called CODONS.

peptidyltransferase

hydrolyze the bond linking the peptide to the tRNA at the A site, causing the nascent protein to be released from the ribosome The binding of these release factors induces peptidyltransferase to hydrolyze the bond linking the peptide to the tRNA at the A site, causing the nascent protein to be released from the ribosome. A third release factor, RF-3 (bound to GTP) then causes the release of RF-1 or RF-2 as GTP is hydrolyzed

Shine-Dalgarno sequence

in E. coli, a purine-rich sequence of nucleotide bases (for example, 5′-UAAGGAGG-3′), is located six to ten bases upstream of the initiating AUG codon on the mRNA molecule— near its 5′-end. (30S ribosome binds, stabilizes ?)

Ribosomes

large complexes of protein and rRNA. They consist of two subunits—one large and one small—whose relative sizes are generally given in terms of their sedimentation coefficients, or S (Svedberg) values. [Note: Because the S values are determined both by shape as well as molecular mass, their numeric values are not strictly additive. For example, the prokaryotic 50S and 30S ribosomal subunits together form a 70S ribosome. The eukaryotic 60S and 40S subunits form an 80S ribosome.] Prokaryotic and eukaryotic ribosomes are similar in structure, and serve the same function, namely, as the "factories" in which the synthesis of proteins occurs.

codon

made up of 3 nucleotide bases in mRNA adenine (A), guanine (G), cytosine (C), uracil (U) (always written from the 5′-end to the 3′-end)

P-site codon

occupied by peptidyl-tRNA. This tRNA carries the chain of amino acids that has already been synthesized. The E site is occupied by the empty tRNA as it is about to exit the ribosome

E-site

occupied by the empty tRNA as it is about to exit the ribosome

universality

specificity of genetic code has been conserved from very early stages of evolution, with only slight differences in the manner in which the code is translated (exception = few codons in mitochondria)

possible combinations

64 different combinations of bases, taken three at a time. 61 of the 64 codons code for the twenty common amino acids. Three of the codons, UAG, UGA, and UAA, do not code for amino acids, but rather are termination codons. When one of these codons appears in an mRNA sequence, it signals synthesis of the protein coded for by that mRNA is completed.

Elongation

Elongation of the polypeptide chain involves the addition of amino acids to the carboxyl end of the growing chain. During elongation, the ribosome moves from the 5′-end to the 3′-end of the mRNA that is being translated. Delivery of the aminoacyl-tRNA whose codon appears next on the mRNA template in the ribosomal A site is facilitated in E. coli by elongation factors EF-Tu and EF-Ts and requires GTP hydrolysis. In eukaryotes, comparable elongation factors are EF-1α and EF-1βγ. Both EF-Ts and EF-1βγ function as nucleotide exchange factors, exchanging their GTP for the guanosine diphosphate (GDP) (from GTP hydrolysis) on EF-Tu and EF-1α. The formation of the peptide bonds is catalyzed by peptidyltransferase, an activity intrinsic to the 23S rRNA found in the 50S ribosomal subunit. Because this rRNA catalyzes the reaction, it is referred to as a ribozyme. After the peptide bond has been formed, the ribosome advances three nucleotides toward the 3′-end of the mRNA. This process is known as translocation and, in prokaryotes, requires the participation of EF-G (eukaryotic cells use EF-2) and GTP hydrolysis. This causes movement of the uncharged tRNA into the ribosomal E site (before being released), and movement of the peptidyl-tRNA into the P site.

termination

Eukaryotes have a single release factor, eRF, which recognizes all three termination codons. A second factor, eRF-3, is thought to function like the prokaryotic RF-3. The newly synthesized polypeptide may undergo further modification as described below, and the ribosomal subunits, mRNA, tRNA, and protein factors can be recycled and used to synthesize another polypeptide.

Initiation

Initiation of protein synthesis involves the assembly of the components of the translation system before peptide bond formation occurs. These components include the two ribosomal subunits, the mRNA to be translated, the aminoacyl-tRNA specified by the first codon in the message, GTP (which provides energy for the process), and initiation factors that facilitate the assembly of this initiation complex. In prokaryotes, three initiation factors are known (IF-1, IF-2, and IF-3), whereas in eukaryotes, there are over ten (designated eIF to indicate eukaryotic origin). Eukaryotes also require ATP for initiation. There are two mechanisms by which the ribosome recognizes the nucleotide sequence that initiates translation: Shine-Dalgarno sequence: In E. coli, a purine-rich sequence of nucleotide bases (for example, 5′-UAAGGAGG-3′), known as the Shine-Dalgarno (SD) sequence, is located six to ten bases upstream of the initiating AUG codon on the mRNA molecule—that is, near its 5′-end. The 16S rRNA component of the 30S ribosomal subunit has a nucleotide sequence near its 3′-end that is complementary to all or part of the SD sequence. Therefore, the mRNA 5′-end and the 3′-end of the 16S ribosomal RNA can form complementary base pairs, thus facilitating the binding and positioning of the 30S ribosomal subunit on the mRNA in close proximity to the initiating AUG codon. Eukaryotic messages do not have SD sequences. In eukaryotes, the 40S ribosomal subunit (aided by members of the elF-4 family of proteins) binds to the cap structure at the 5′-end of the mRNA and moves down the mRNA until it encounters the initiator AUG. This "scanning" process requires ATP.

Wobble hypothesis

The mechanism by which tRNAs can recognize more than one codon for a specific amino acid is described by the "wobble" hypothesis in which the base at the 5′-end of the anticodon (the "first" base of the anticodon) is not as spatially defined as the other two bases. Movement of that first base allows nontraditional base-pairing with the 3′-base of the codon (the "last" base of the codon). This movement is called "wobble" and allows a single tRNA to recognize more than one codon. The result of wobbling is that there need not be 61 tRNA species to read the 61 codons coding for amino acids.

Steps in Protein Synthesis

The pathway of protein synthesis translates the three-letter alphabet of nucleotide sequences on mRNA into the twenty-letter alphabet of amino acids that constitute proteins. The mRNA is translated from its 5′-end to its 3′-end, producing a protein synthesized from its amino-terminal end to its carboxyl-terminal end. Prokaryotic mRNA often have several coding regions, that is, they are polycistronic. Each coding region has its own initiation and termination codon and produces a separate species of polypeptide. In contrast, each eukaryotic mRNA has only one coding region, i.e., it is monocistronic. The process of translation is divided into three separate steps: initiation, elongation, and termination. The polypeptide chains produced may be modified by posttranslational modification. Eukaryotic protein synthesis resembles that of prokaryotes in most details.

stop codons

UAG, UGA, and UAA RF-1, which recognizes the termination codons UAA and UAG, and RF-2, which recognizes UGA and UAA.

stop codons

UAG, UGA, and UAA,

specificity

a particular codon ALWAYS codes for the same amino acid ex: AUG is always methionine

eIF

eukaryotic initiation factor

A, P, and E sites on the ribosome

three binding sites for tRNA molecules each of which extends over both subunits. Together, they cover three neighboring codons.

If one amino acid is missing (ex: in diet)

translation stops at the codon specifying that amino acid.

Ricin (from castor beans)

very potent toxin that exerts its effects by removing an adenine from 28S ribosomal RNA, thus inhibiting eukaryotic ribosomes.