Do 1 of the following 3. You are studying a new operon in E. colí involved in ph

Question

Do 1 of the following 3. You are studying a new operon in E. colí involved in phenylalanine biosynthesis. (a) How would you predict this operon is regulated (eg, inducible or repressible by phenylalanine. positively or negatively, etc)? Why? (b) You sequence the operon and discover that it contains a short open reading frame near the 5'-end of the operon that contains several codons for phenylalanine. What prediction would you make about the function of this leader sequence and the peptide it encodes? (e) What is this kind of regulation called and would it work in a eukaryotic cell? Why or why not? 4. (a) Describe the important features of initiation, clongation and termination stages of translation process. (b) Compare and contrast these translational processes in prokaryotes and eukaryotes, summarize the major differences (such as enzymes, protein factors, DNA sequence elements, RNAs, and/or factors that you believe are involved)

Explanation / Answer

4.

Initiation of Translation

Protein synthesis begins with the formation of an initiation complex. In E. coli, this complex involves the small 30S ribosome, the mRNA template, three initiation factors (IFs; IF-1, IF-2, and IF-3), and a special initiator tRNA, called. The initiator tRNA interacts with the start codon AUG (or rarely, GUG), links to a formylated methionine called fMet, and can also bind IF-2. When an in-frame AUG is encountered during translation elongation, a non-formylated methionine is inserted by a regular Met-tRNAMet.

In E. coli mRNA, a sequence upstream of the first AUG codon, called the Shine-Dalgarno sequence (AGGAGG), interacts with the rRNA molecules that compose the ribosome. This interaction anchors the 30S ribosomal subunit at the correct location on the mRNA template. Guanosine triphosphate (GTP), which is a purine nucleotide triphosphate, acts as an energy source during translation—both at the start of elongation and during the ribosome’s translocation.

In eukaryotes, a similar initiation complex forms, comprising mRNA, the 40S small ribosomal subunit, IFs, and nucleoside triphosphates (GTP and ATP). The charged initiator tRNA, called Met-tRNAi, does not bind fMet in eukaryotes, but is distinct from other Met-tRNAs in that it can bind IFs.

Instead of depositing at the Shine-Dalgarno sequence, the eukaryotic initiation complex recognizes the 7-methylguanosine cap at the 5? end of the mRNA. A cap-binding protein (CBP) and several other IFs assist the movement of the ribosome to the 5? cap. Once at the cap, the initiation complex tracks along the mRNA in the 5? to 3? direction, searching for the AUG start codon. Many eukaryotic mRNAs are translated from the first AUG, but this is not always the case. According to Kozak’s rules, the nucleotides around the AUG indicate whether it is the correct start codon. Kozak’s rules state that the following consensus sequence must appear around the AUG of vertebrate genes: 5?-gccRccAUGG-3?. The R (for purine) indicates a site that can be either A or G, but cannot be C or U. Essentially, the closer the sequence is to this consensus, the higher the efficiency of translation.

Once the appropriate AUG is identified, the other proteins and CBP dissociate, and the 60S subunit binds to the complex of Met-tRNAi, mRNA, and the 40S subunit. This step completes the initiation of translation in eukaryotes.

Translation, Elongation, and Termination

In prokaryotes and eukaryotes, the basics of elongation are the same, so we will review elongation from the perspective of E. coli. The 50S ribosomal subunit of E. coli consists of three compartments: the A (aminoacyl) site binds incoming charged aminoacyl tRNAs. The P (peptidyl) site binds charged tRNAs carrying amino acids that have formed peptide bonds with the growing polypeptide chain but have not yet dissociated from their corresponding tRNA. The E (exit) site releases dissociated tRNAs so that they can be recharged with free amino acids. Similarly, the eukaryotic Met-tRNAi, with help from other proteins of the initiation complex, binds directly to the P site. In both cases, this creates an initiation complex with a free A site ready to accept the tRNA corresponding to the first codon after the AUG.

During translation elongation, the mRNA template provides specificity. As the ribosome moves along the mRNA, each mRNA codon comes into register, and specific binding with the corresponding charged tRNA anticodon is ensured. If mRNA were not present in the elongation complex, the ribosome would bind tRNAs nonspecifically.

Elongation proceeds with charged tRNAs entering the A site and then shifting to the P site followed by the E site with each single-codon “step” of the ribosome. Ribosomal steps are induced by conformational changes that advance the ribosome by three bases in the 3? direction. The energy for each step of the ribosome is donated by an elongation factor that hydrolyzes GTP. Peptide bonds form between the amino group of the amino acid attached to the A-site tRNA and the carboxyl group of the amino acid attached to the P-site tRNA. The formation of each peptide bond is catalyzed by peptidyl transferase, an RNA-based enzyme that is integrated into the 50S ribosomal subunit. The energy for each peptide bond formation is derived from GTP hydrolysis, which is catalyzed by a separate elongation factor. The amino acid bound to the P-site tRNA is also linked to the growing polypeptide chain. As the ribosome steps across the mRNA, the former P-site tRNA enters the E site, detaches from the amino acid, and is expelled. Amazingly, the E. coli translation apparatus takes only 0.05 seconds to add each amino acid, meaning that a 200-amino acid protein can be translated in just 10 seconds.

Termination of translation occurs when a nonsense codon (UAA, UAG, or UGA) is encountered. Upon aligning with the A site, these nonsense codons are recognized by release factors in prokaryotes and eukaryotes that instruct peptidyl transferase to add a water molecule to the carboxyl end of the P-site amino acid. This reaction forces the P-site amino acid to detach from its tRNA, and the newly made protein is released. The small and large ribosomal subunits dissociate from the mRNA and from each other; they are recruited almost immediately into another translation initiation complex. After many ribosomes have completed translation, the mRNA is degraded so the nucleotides can be reused in another transcription reaction.

b)

Translation

Prokaryotes

Eukaryotes

Initiation factor

Three (IF3, IF2, IF1)

Nine (eIF4F complex; eIF4E, eIF4G,eIF4A)

Ribosome

30S and 50S

40S and 60s

Shine-Dalgarno sequence at 5’ end

Present (5’ UAAGGAGG3’).

absent

Poly A tail at 3’end

absent

present

Elongation factor

EF-TU, EF-TS, EF-G-GTP

eEF1?, eEF1??,eEF2

Release factors

Three (RF1, RF2 and RF3)

One (eRF)

Chain initiating amino acid

N-formyl methionine

methionine

mRNA

Polycistronic

Monocistonic

Stability of mRNA

Unstable

Quite stable

The rate of translation

Faster process; adds 20 amino acids per second

Slower process; adds one amino acid per second

Placement of AUG codon P-site

By shine dalgarno sequence

Scanning of mRNA

Translation

Prokaryotes

Eukaryotes

Initiation factor

Three (IF3, IF2, IF1)

Nine (eIF4F complex; eIF4E, eIF4G,eIF4A)

Ribosome

30S and 50S

40S and 60s

Shine-Dalgarno sequence at 5’ end

Present (5’ UAAGGAGG3’).

absent

Poly A tail at 3’end

absent

present

Elongation factor

EF-TU, EF-TS, EF-G-GTP

eEF1?, eEF1??,eEF2

Release factors

Three (RF1, RF2 and RF3)

One (eRF)

Chain initiating amino acid

N-formyl methionine

methionine

mRNA

Polycistronic

Monocistonic

Stability of mRNA

Unstable

Quite stable

The rate of translation

Faster process; adds 20 amino acids per second

Slower process; adds one amino acid per second

Placement of AUG codon P-site

By shine dalgarno sequence

Scanning of mRNA

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