Mrs. Ofelia Solano Saludar

Department of Natural Sciences University of St. La Salle

TRANSLATION

Transcription, RNA

processing, and translation

are the processes that

link DNA sequences to the synthesis of a specific polypeptide

chain.

Translation is a well conserved process among prokaryotes and eukaryotes.

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.

Additional processing and assembly is often required to modify the proteins.

TRANSLATION

1.Ribosomes Each ribosome has a large and a small subunit. These are composed of proteins and rRNA, the

most abundant RNA in the cell. rRNA is the main constituent at the interphase

between the two subunits and of the A and P sites.

It is the catalyst for peptide bond formation. After rRNA genes are transcribed to rRNA in the

nucleus, the rRNA and proteins form the subunits in the nucleolus. The subunits exit the nucleus via nuclear pores.

The large and small subunits join to form a functional ribosome only when they attach to an mRNA molecule.

THE TOOLS OF TRANSLATION

A functional ribosome has 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’ end of the mRNA, placing the mRNA in the proper position for the translation of its first codon.

The binding sites are located at or near the interface between the large and small subunits.

The P site holds the tRNA carrying the growing polypeptide chain.

The A site carries the tRNA with the next amino acid.

Discharged tRNAs leave the ribosome at the E site.

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2.tRNA molecules consists of a strand of about 80 nucleotides that folds back on itself to form a 3D structure. It contains: 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).

Structure of tRNAs. (a) Although the exact nucleotide sequence varies among tRNAs, they all fold into 4 base-paired stems and 3 loops. The

CCAsequence at the 3’ end also is found in all tRNAs. Attachment of an

amino acid to the 3’ A yields an aminoacyl-tRNA. Dihydrouridine (D) is present in the D loop; ribothymidine (T) and pseudouridine (Ý) are in the TCG loop. The triplet at the tip of the anti-codon loop base-pairs with the

corresponding codon in mRNA. (b) 3-D model of the generalized backbone of all tRNAs.

3.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).

Nonstandard codon-anticodon base pairing at the wobble position.

The base in the 3rd (or wobble) position of an

mRNA codon often forms a nonstandard base pair with the base in the 1st position

of a tRNA anticodon. Wobble pairing allows a tRNA to recognize more than one mRNA codon

(top); it allows a codon to be recognized by more than one kind of tRNA

(bottom), although each tRNA will bear the same

amino acid. A tRNA with I (inosine) in the wobble position can “read” 3

codons, and a tRNA with G or U in the wobble position

can read 2 codons.

Rules for base pairing between the 3rd base of the codon and

anticodon are relaxed (wobble).

A is possible in the wobble position of the anticodon, but it

is almost never found in nature.

Aminoacyl-tRNA synthetases catalyzes the formation of an ester bond between the carboxyl group of an amino acid and the 3’ OH group of the appropriate tRNA. 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. The tRNA covalently bonds to the amino acid to displace the AMP and the aminoacyl tRNA is then released from the

enzyme.

All 3 phase of translation (initiation, elongation, termination) require protein “factors” that aid in the translation process. Both initiation and chain elongation require energy provided by the hydrolysis of GTP.

THE PROCESS OF TRANSLATION

Formation of the prokaryotic translation initiation

complex Three initiation factors (IF 1,

2, 3) and GTP bind to the small ribosomal subunit.

The mRNA-binding site to the ribosome is composed of a

portion of the 16S rRNA of the small ribosomal subunit. The 3’

end of the 16S rRNA bears a pyrimidine-rich stretch that base pairs with the Shine-Dalgarno sequence of the

mRNA.

The resulting 70S initiation complex has

fMet-tRNA-fMet (N-formyl-methionine)

residing in the ribosome's P site.

The large ribosomal subunit joins the

complex.

The Shine- Dalgarno sequence in E. coli is AGGAGGU, which helps recruit the ribosome to the mRNA to initiate protein synthesis by aligning it with the start codon.

The complementary sequence (UCCUCC) is located at the 3' end of the 16S rRNA in the ribosome.

Mutations in the Shine-Dalgarno sequence can reduce translation. This reduction is due to a reduced mRNA-ribosome pairing efficiency, as evidenced by the fact that complementary mutations in the anti-Shine-Dalgarno sequence can restore translation.

When the Shine-Dalgarno sequence and the anti-Shine-Dalgarno sequence pair, the translation initiation factors IF2-GTP, IF1, IF3, as well as the initiator tRNA fMet-tRNA(fmet) are recruited to the ribosome.

The eukaryotic equivalent of the Shine-Dalgarno sequence is called the Kozak sequence.

1. Elongation begins with the binding of the 2nd aminoacyl tRNA at the 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 help recycle the EF-Tu.

2. A peptide bond is formed between the carboxyl group of the terminal amino acid 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. The next mRNA codon is moved

into the A site, which is open for the next aminoacyl

tRNA. 4. These events are repeated

for each additional amino acid.

ELONGATION

TERMINATION

Termination of protein synthesis depends on release factors that recognize the 3 stop codons.

When a stop arrives at the A site, it is recognized and bound by a protein release factor (RF1 = UAA or UAG RF2 = UAA or UGA RF3 = a GTPase like EF-Tu and binds in a similar A-site location).

This RF causes the poly-peptide to be transferred to a molecule of H2O to cause its release from the tRNA and the dissociation of the other components of the elongation complex.

Typically a single mRNA is used to make many copies of a polypeptide simultaneously.

Multiple ribosomes, polyribosomes, may trail along the same mRNA.

A ribosome requires less than a minute to translate an average-sized mRNA into a polypeptide.

A ribosome complexes with mRNA and an activated

initiator tRNA, at the start codon. The Kozak

consensus sequence, gccRccAUGG, (R is a purine 3 bases upstream of the AUG),

is recognized by the ribosome as the translational

start site. Large and small ribosomal subunits not

actively engaged in translation are kept apart by

binding of 2 initiation factors, designated eIF3 and eIF6 in eukaryotes. A translation pre-initiation

complex is formed when the 40S subunit–eIF3 complex is

bound by eIF1A and a ternary complex of the

MettRNAi Met, eIF2, and GTP

EUKARYOTIC TRANSLATION

Initiation of translation in eukaryotes. When a ribosome dissociates at

the termination oftranslation, the 40S and 60S subunits associate

with initiationfactors eIF3 and eIF6,

forming complexes that can initiate another round

of translation. (1) and (2) Sequential

addition of the indicated components to the 40S subunit–eIF3 complex

forms the initiation complex.

(3) Scanning of the mRNA by the

associated initiation complex leads to positioning of the small subunit and

bound Met-tRNAi Met at the start codon.

(4) Association of the large subunit (60S)

forms an 80S ribosome ready to

translate the mRNA. Two initiation factors,

eIF5 and eIF6 are GTP-binding proteins, whose bound GTP is hydrolyzed during

translation initiation.

Cycle of peptidyl chain elongation during

translation in eukaryotes.

Once the 80S ribosome with Met-tRNAi Met in the

ribosome P site is assembled (top), aternary complex

bearing the 2nd amino acid (aa2) coded by the

mRNA binds to the A site (1), followed by a

conformational change in the ribosome induced by hydrolysis of GTP in

EF1-GTP (2).

The large rRNA catalyzes peptide bond formation between Meti and aa2 (3). Hydrolysis of

GTP in EF2-GTP causes change in the

ribosome that results in its translocation one codon along

the mRNA and shifts the unacylated tRNAi Met to the E site

and the tRNA with the bound peptide to the P site (4). The cycle can begin again with

binding of a ternary complex bearing aa3 to the now-open A site. In the 2nd and subsequent elongation cycles, the tRNA at the E site is ejected during (2)

as a result of the conformational change induced by hydrolysis of

GTP in EF1-GTP.

Termination of translation in eukaryotes.

When a ribosome bearing a nascent protein chain

reaches a stop codon (UAA, UGA, UAG), release factor eRF1 enters the ribosomal

complex, probably at or near the A site together with

eRF3-GTP. Hydrolysis of the bound GTP is accompanied by cleavage of the peptide

chain from the tRNA in the P site and

release of the tRNAs and the two ribosomal subunits.

Model of protein synthesis on circular polysomes and recycling of ribosomal subunits. Multiple individual ribosomes can

simultaneously translate a eukaryotic mRNA, shown here in circular form stabilized by interactions between proteins bound at the 3’ and 5’ ends. When a ribosome completes translation and dissociates from the 3’ end, the separated subunits can rapidly find the nearby 5’ cap (m7G)

and initiate another round of synthesis.

Overview of protein structure and function.

(a) The linear sequence of amino acids (10 structure) folds into helices or sheets (20 structure) which pack into a globular or fibrous

domain (30 structure). Some individual proteins

self-associate into complexes (40 structure).

(b) Proteins display functions that arise from

specific binding interactions and conformational

changes in the structure of a properly folded protein.

PROTEIN STRUCTURE, TARGETING AND SORTING.pptx

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