bc21c translation

7/27/2019 BC21C Translation

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Gene Expression:Translation

Paul D. Brown, [email protected]

BC21C: Molecular Biology I

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Learning Objectives Describe the process of translation and

antibiotic inhibitors. Briefly describe the function of the

following: 30S and 50S ribosomal subunits ribosome binding site start codon and nonsense (stop) codon initiation complex tRNA and aminoacyl-tRNA anticodon P-site of ribosome A-site of ribosome peptidyl transferase release factors


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Translation: the RNA-directed synthesisof a polypeptide: a closer look

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Correct Reading Frame is Critical


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A tRNA molecule consists of a strand ofabout 80 nucleotides that folds back on

itself to form a three-dimensional structure. It includes a loop containing the anticodon andan attachment site at the 3 end for an aminoacid.

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Translation can be divided into four stages: Activation

Initiation

Elongation

Termination

Initiation, elongation and termination

require protein factors that aid in the

translation process.

Activation require energy from ATP andboth initiation and chain elongation require

energy from GTP.


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Slide 7

Each amino acid isjoined to the correct

tRNA by aminoacyl-tRNA synthetase.

The 20 differentsynthetases match the20 different amino acids. Each has active sites for

only a specific tRNA andamino acid combination.

The synthetase catalyzesa covalent bond between

them, forming aminoacyl-tRNA or activated aminoacid.

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Initiation brings together mRNA, a tRNAwith the first amino acid, and the tworibosomal subunits (with rRNA).


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ProkaryotesEukaryotes

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Ribosomes facilitate the specific coupling of

the tRNA anticodons with mRNA codons. Each ribosome has a large and a small

subunit. These are composed of proteins and ribosomal

RNA (rRNA), the most abundant RNA in thecell.


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Elongation consists of a series of three

step cycles as each amino acid is added tothe proceeding one.

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Recent advances in our understanding of

the structure of the ribosome stronglysupports the hypothesis that rRNA, notprotein, carries out the ribosomesfunctions. RNA is the main constituent at the interphase

between the two subunits and the A and Psites.

It is the catalyst forpeptide bond formation


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During translocation, the ribosome movesthe tRNA with the attached polypeptide

from the A site to the P site.

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Termination occurs when one of the threestop codons reaches the A site.


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

Multiple ribosomes, polyribosomes , may trailalong the same mRNA.

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

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During and after synthesis, a polypeptide coilsand folds to its three-dimensional shapespontaneously. The primary structure, the order of amino acids,

determines the secondary and tertiary structure.

Chaperone proteins may aid correct folding.

In addition, proteins may require posttranslationalmodifications before doing their particular job. This may require additions like sugars, lipids, or

phosphate groups to amino acids.

Enzymes may remove some amino acids or cleavewhole polypeptide chains.

Two or more polypeptides may join to form a protein.


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The Case of Insulin

Polypeptide hormone

Synthesized in the -cells of the pancreas

Consists of two polypeptide chains A and B

Synthesized as a single polypeptide chain110 aa residues -preproinsulin 24-residue signal peptide attached to an 86-

residue -proinsulin

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Processing of insulin

Preproinsulin directed to ER

Initiating Met is removed

Signal peptide removed signal peptidase

Cysteine residues form disulfide bonds

Internal C-peptide removed

Removal of basic residues yields maturehormone


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Post-translational modif ication

Glycosylation Carbohydrate bound to proteins, occurs in ER &

Golgi

Methylation Specific lysines undergo N-methylation

Phosphorylation Protein kinases

Sulfation

Addition of sulfate to tyrosyl hydroxyl groups,e.g., in fibrinogen

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The cellular machinery of proteinsynthesis and ER targeting isdominated by various kinds of RNA.

DNA may be the genetic material of allliving cells today, but RNA is muchmore versatile.

RNA plays multiple roles in the cell: areview


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The diversefunctions of

RNA rangefrom structuraltoinformationalto catalytic.

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Although bacteria and eukaryotes carry outtranscription and translation in very similarways, they do have differences in cellularmachinery and in details of the processes. Eukaryotic RNA polymerases differ from those of

prokaryotes and require transcription factors. They differ in how transcription is terminated,

and how translation is initiated Their ribosomes are also different.

Comparing protein synthesis inprokaryotes and eukaryotes: a review


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One majordifference:

prokaryotescan transcribeand translatethe same genesimultaneously.

The newprotein quicklydiffuses to itsoperating site.

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In eukaryotes, the nuclear envelopesegregates transcription from translation.

In addition, extensive RNA processing isinserted between these processes. This provides additional steps whose

regulation helps coordinate the elaborateactivities of a eukaryotic cell.

In addition, eukaryotic cells havecomplicated mechanisms for targeting

proteins to the appropriate point of need.


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Antibiotic Inhibitors ofTranslation

Bind to 30S subunit

Inhibits initiation and causesmisreading of mRNA

Streptomycin

Bind to 70S subunit

Prevent dissociation of 70Sribosomes; no translation

Neomycin,Kanamycin

Bind to 50S ribosomal subunit

Inhibit peptide bond formation(translocation)

Gentamicin,Erythromycin,LincomycinChloramphenicol

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Antibiotic Inhibitors ofTranslation

Inhibits translocation in eukaryotesCycloheximideIrreversibly inactivates eukaryoticribosomes by depurinating an A in 28SrRNA

Ricin

Causes premature chain termination byacting as analog of aa-tRNA (bothprokaryotes & eukaryotes)

Puromycin

Bind to 30S subunitPrevents binding of aa-tRNA to mRNAribosome complex

Tetracycline

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