Chapter 6Gene Expression: Translation

Copyright © 2010 Pearson Education Inc.

Chemical Structure of Proteins◦ Proteins are built from amino acids held together by

peptide bonds. The amino acids confer shape and properties to the protein.

◦ Two or more polypeptide chains may associate to form a protein complex. Each cell type has characteristic proteins associated with its function.

‑A carboxyl group (COOH), which is also usually charged at cellular pH (COO-).

A hydrogen atom (H). ‑An R group, which is

different for each amino acid and confers distinctive properties. The R groups in an amino acid chain give polypeptides their structural and functional properties.

All amino acids (except proline) have a common structure.

The -carbon is bonded to: ‑An amino group (NH2), which is usually charged at

cellular pH (NH3+).

Polypeptides are chains of amino acids joined by covalent peptide bonds. A peptide bond forms between the carboxyl group of one amino acid and the amino group of another

Polypeptides are un-branched and have a free amino group at one end (the N terminus) and a carboxyl group at the other (the C terminus). The N-terminal end defines the beginning of the polypeptide.

Proteins have up to four levels of organization:◦ Primary structure is the amino

acid sequence of the polypeptide. This is determined by the nucleotide sequence of the corresponding gene.

◦ Secondary structure is folding and twisting of regions within a polypeptide, resulting from electrostatic attractions and/or hydrogen bonding. Common examples are the -helix and -pleated sheet.

Tertiary structure is the three-dimensional shape of a single polypeptide chain, conformation.◦ It is the biologically functional

form Quaternary structure

occurs in multi-subunit proteins, as a result of the association of polypeptide chains.

4 nucleotides code for 20 amino acids; How?◦ Combinations◦ How many combinations?◦ How is done?

Evidence for a triplet code came from experiments in bacteriophage T4. A virulent phage, T4 produces 100–200 progeny phage per infected E. coli cell and produces plaques on a “lawn” of E. coli.

A mutant T4 phage strain called rII can be identified in two ways:◦ a. T4 phage rII mutants produce clear plaques

when grown on E. coli strain B, while the wild-type r+ phage make turbid plaques on E. coli B.

◦ b. T4 phage rII mutants do not grow in E. coli strain K-12(l), while r+ T4 phage do.

The rII mutant strain was produced by treating r+ phage with proflavin. Proflavin causes frameshift mutations by inserting or deleting base pairs of DNA.

The reasoning was that reversion of a deletion (a -mutation) could be caused by a nearby insertion (a +mutation), and vice versa.

Revertants of rII to r+ can be detected by plaques on E. coli K-12(l).

Combine genetically distinct rII mutants of the same type (either all - or all +), and only when it is a combination of three (or multiple of three) are there high levels of reversion.

This indicates that the genetic code is a triplet code.

Nirenberg and Khorana (1968) using cell-free, protein-synthesizing systems from E. coli that included ribosomes and required protein factors, along with tRNAs carrying radiolabeled amino acids.

Synthetic mRNAs were used in the cell-free translation system, and the resulting polypeptides analyzed:◦ a. When the mRNA contained one type of base, the results

were clear [e.g., poly(U) was responsible for a chain of phenylalanines].

◦ b. Synthetic random copolymers of mRNA (a mix of two different nucleotides, A and C for example) can contain eight possible codons, including two with only one nucleotide (e.g., AAA and CCC) whose amino acid is already known. By altering the concentrations of the two nucleotides and analyzing the polypeptides produced, the codons can be deduced.

◦ c. Copolymers with a known repeating sequence (e.g., UCUCUCUCU) will produce polypeptides with alternating amino acids (e.g., leu-ser-leu-ser), indicating that UCU is one and CUC is the other, but not which is which.

It is a triplet code. It is comma free. The mRNA is

read continuously, three bases at a time, without skipping any bases.

It is nonoverlapping. Each nucleotide is part of only one codon and is read only once during translation.

It is almost universal, most codons have the same amino acid meaning. (Protozoan Tetrahymena and mitochondria of some organisms).

It is degenerate. Of 20 amino acids, 18 are encoded by more than one codon. Met (AUG) and Trp (UGG) are the exceptions.

The code has start and stop signals. AUG is the usual start defines the open reading frame. Generally three stop codons: UAG (amber), UAA (ochre), and UGA (opal).

Wobble occurs in the anticodon.

Ribosomes translate the genetic message of mRNA into proteins.

The mRNA is translated 5’ to 3’, producing a corresponding N-terminal-to-C-terminal polypeptide.

Amino acids are bound to tRNAs used by the ribosome to build proteins.

Amino acids are inserted into the polypeptide in the proper sequence due to:◦ a. Specific binding of each amino acid to its

tRNA.◦ b. Specific base-pairing between the mRNA

codon and tRNA anticodon.

The tRNAs are 75–90 nt in length, and each tRNA has a different sequence.

All tRNAs can be shown in a cloverleaf structure, with complementary base pairing between regions to form four stems and loops. Loop II contains the anticodon.

All tRNAs have CCA (added posttranscriptionally) at their 3’ ends, for the amino attachement.

Extensive chemical modifications are performed on all tRNAs after transcription.

Aminoacyl-tRNA synthetase attaches amino acids to their specific tRNA molecules. The charging process (aminoacylation) produces a charged tRNA (aminoacyl-tRNA), using energy from ATP hydrolysis. There are 20 different aminoacyl-tRNA synthetase enzymes, one for each amino acid, and each enzyme recognizes the structure of the specific tRNA(s) it charges.

The amino acid and ATP bind to the specific aminoacyl-tRNA synthetase enzyme.

The tRNA binds to the enzyme, and the amino acid is transferred onto it, displacing the AMP. The aminoacyl-tRNA is released from the enzyme.

The amino acid is covalently attached by its carboxyl group to the 3’ end of the tRNA. Every tRNA has a 3’ adenine, and the amino acid is attached to the 3’-OH or 2’-OH of this nucleotide

Ribosomes are the catalyst for protein synthesis, facilitating binding of charged tRNAs to the mRNA so that peptide bonds can form.

Ribosomes in both bacteria and eukaryotes consist of two subunits of unequal size (large and small), each with at least one rRNA and many ribosomal proteins◦ a. Bacterial ribosomes are 70S, with 50S and 30S

subunits. i. The 50S subunit contains the 23S rRNA and 5S rRNA. Ii. The 30S subunit contains the 16S rRNA.

◦ b. Mammalian ribosomes are 80S, with 60S and 40S subunits. i. The 60S subunit contains the 28S rRNA, the 5.8S rRNA, and the

5S. ii. The 40S subunit contains the 18S rRNA.

When translating an mRNA, specific sites on the ribosome perform specific functions:◦ a. The A site binds incoming

aminoacyl-tRNAs.◦ b. The P site contains the tRNA

carrying the growing polypeptide chain.

◦ c. The E site allows exit of the tRNA after donating its amino acid.

The P and A sites consist of regions of both subunits of the ribosome, while E is a region of the large subunit.

DNA regions that encode rRNA are called ribosomal DNA (rDNA) or rRNA transcription units.

E. coli is a typical bacterium, with seven rRNA coding regions scattered in its chromosome.◦ a. Each rRNA transcription unit has a single promoter and

contains the genes 16S-23S-5S, in that order, with non-rRNA sequences as spacers.

◦ b. The pre-rRNA transcript associates with ribosomal proteins and is cleaved by RNases to release the three rRNAs. These rRNAs associate with ribosomal proteins to form functional ribosomal subunits.

Eukaryotes generally have many copies of the rRNA genes.◦ a. The three rRNA genes with homology to prokaryotic rRNA

genes are 18S-5.8S-28S, in that order. In the chromosome these genes are tandemly repeated 100–1,000 times to form rDNA repeat units.

◦ b. A nucleolus forms around each rDNA repeat unit and then they fuse to make one nucleolus. Ribosomal subunits are produced in this structure by addition of the 5S rRNA and ribosomal proteins.

◦ c. RNA polymerase I transcribes the rDNA repeat units, producing a pre-rRNA molecule containing the 18S, 5.8S, and 28S rRNAs, separated by spacer sequences.

◦ d. The 5S rRNA gene copies are located elsewhere in the genome and are transcribed by RNA polymerase III.

Protein synthesis is similar in bacteria and eukaryotes. Some significant differences will be discussed.

In both, translation is divided into three stages:◦ a. Initiation.◦ b. Elongation.◦ c. Termination.

Initiation of translation requires:◦ a. An mRNA.◦ b. A ribosome.◦ c. A specific initiator tRNA.◦ d. Initiation factors (IF).◦ e. GTP.

Bacterial translation begins with binding of the 30S ribosomal subunit complexed with IF-1 and IF-3 to mRNA near the AUG codon.

Ribosome binding to mRNA requires more than the AUG:◦ a. The ribosome binds at a

ribosome-binding site (RBS), 8–12 nucleotides upstream from the AUG, where it is oriented to the correct reading frame for protein synthesis.

◦ b. Discovered by Shine and Dalgarno, these purine-rich sequences (AGGAG) are complementary to the 3’ end of the 16S rRNA.

◦ c. Further mutational analysis shows that complementarity between the Shine–Dalgarno sequence and the 3’ end of 16S rRNA is important in ribosome binding to the mRNA.

Initiator tRNA binds the AUG to which the 30S subunit is bound. AUG universally encodes methionine. ◦ a. Initiator methionine in

bacteria is formylmethionine (fMet). It is carried by a specific tRNA (with the anticodon 5’-CAU-3’).

◦ b. The tRNA first binds a methionine, and then transformylase attaches a formyl group to the methionine, making fMet-tRNA.fMET (a charged initiator tRNA).

◦ c. Methionines at sites other than the beginning of a polypeptide are inserted by tRNA.Met (a different tRNA), which is charged by the same aminoacyl-tRNA synthetase as tRNA.fMet.

Met-tRNA.fMet is brought to the 30S-mRNA complex by IF-2/GTP.

IF-1 blocks the A site on the 30S subunit, so only P site is open for the tRNA.fMet, forming the 30S initiation complex.

The 50S ribosomal subunit binds the complex, GTP is hydrolyzed, and the three IF are released, forming a 70S initiation complex.

Initiation of translation is similar in eukaryotes but more complex. Initiation factors are called eIF. Main differences in eukaryotic translation:

a. Initiator methionine is not modified. As in prokaryotes, it is attached to a special tRNA.

b. Ribosome binding involves the 5’ cap, not a Shine–Dalgarno sequence.◦ i.‑Eukaryotic initiator factor (eIF-4F) is a protein

multimer, including cap-binding protein (CBP). It binds the 5’ mRNA cap.

◦ ii.‑Then the 40S subunit, complexed with initiator Met-tRNA, several eIFs, and GTP, binds the cap complex, including other eIFs.

◦ iii.‑The initiator complex scans the mRNA for a Kozak sequence including the (first) AUG start codon.

◦ iv.‑Once there, 40S binds and then 60S binds, displacing the eIFs and creating the 80S initiation complex with initiator Met-tRNA in the ribosome’s P site. c. The eukaryotic mRNA’s 3’ poly(A) tail also interacts with the 5’ cap.

Poly(A) binding protein (PABP) binds the poly(A) and binds a protein in eIF-4F on the cap, circularizing the mRNA and stimulating translation.

Elongation of the amino acid chain has three steps:◦ a. Binding of aminoacyl-

tRNA to the ribosome.◦ b. Formation of a peptide

bond.◦ c. Translocation of the

ribosome to the next codon.

Protein synthesis begins with fMet-tRNA in the P site of the ribosome. The next charged tRNA approaches the ribosome bound to EF-Tu-GTP. When the charged tRNA hydrogen bonds with the codon in the ribosome’s A site, hydrolysis of GTP releases EF-Tu-GDP.

EF-Tu is recycled with assistance from EF-Ts, which removes the GDP and replaces it with GTP, preparing EF-Tu-GTP to escort another aminoacyl tRNA to the ribosome.

The process is similar in eukaryotes, with eEF-1A taking the place of EF-Tu, and eEF-1B the role of EF-Ts.

The two aminoacyl-tRNAs are positioned by the ribosome for peptide bond formation, which occurs in two steps:◦ a. In the P site, the bond between the

amino acid and its tRNA is cleaved.◦ b. Peptidyl transferase forms a

peptide bond between the now-free amino acid in the P site and the amino acid attached to the tRNA in the A site. 23S rRNA is most likely the catalyst.

◦ c. The tRNA in the A site now has the growing polypeptide chain attached to it.

The ribosome now advances one codon along the mRNA. EF-G is used in translocation in prokaryotes. EF-G-GTP binds the ribosome, GTP is hydrolyzed, and the ribosome moves one codon while the uncharged tRNA leaves the P site. In eukaryotes factor is called eEF-2.

Release of the uncharged tRNA involves the 50S ribosomal E (for Exit) site. Binding to the A site is blocked until tRNA is released at the E site.

During translocation the peptidyl-tRNA remains attached to its codon but is transferred from the ribosomal A site to the P site. This allows release of the uncharged tRNA and EF-G, which will be reused.

The vacant A site contains a new codon, and an aminoacyl-tRNA with the correct anticodon can enter and bind.

In both bacteria and eukaryotes, simultaneous translation occurs creating a polyribosome (polysome) that efficiently produces many polypeptides.

Termination is signaled

by a stop codon (UAA, UAG, UGA) that has no corresponding tRNA.

Release factors (RF) assist the ribosome in recognizing the stop codon and terminating translation.

In E. coli:◦ i. RF1 recognizes UAA and UAG.◦ ii. RF2 recognizes UAA and UGA.◦ iii. RF3 stimulates termination via

GTP hydrolysis.◦ Iv.‑RRF (ribosome recycling

factor) binds the A site, EF-G translocates the ribosome, RRF then releases the last uncharged tRNA and EF-G releases RRF, causing the ribosomal subunits to dissociate from the mRNA.

In eukaryotes, eRF1 recognizes all three stop codons, while eRF3 stimulates termination. Ribosome recycling occurs without an equivalent of RRF

Both bacteria and eukaryotes secrete proteins, and eukaryotes must move proteins into intracellular compartments. Signal sequences on the proteins direct them to their destinations.

Blobel et al. (1975) showed that localization of the new protein results from hydrophobic signal (leader) sequences in the polypeptide.

In eukaryotes, proteins synthesized on the rough ER (endoplasmic reticulum) are glycosylated and then transported in vesicles to the Golgi apparatus. The Golgi sorts proteins based on their signals and then sends them to their destinations.

The required signal sequence for a protein to enter the ER is 15–30 N-terminal amino acids.

As the signal sequence is produced by translation, it is bound by a signal recognition particle (SRP) composed of RNA and protein.

The SRP suspends translation until the complex binds a docking protein on the ER membrane.

Once bound the signal sequence is inserted into the membrane, SRP is released, and translation resumes. Synthesis through the membrane into the ER, in an example of cotranslational transport.

In the ER cisternal space, the signal sequence is removed by signal peptidase. The protein is usually glycosylated and then transferred to the Golgi for sorting.