B M B 400, Part Three

Gene Expression and Protein Synthesis

Section V = Chapter 14

TRANSLATION

A reminder: mRNA encodes the polypeptide with each amino acid designated by a string of three nucleotides. tRNAs serve as the adaptors to translate from the language of nucleic acids to that of proteins. Ribosomes are the factories for protein synthesis.

Figure 3.5.1.

A. tRNAs

1. The transfer RNAs, or tRNAs serve as adaptors to align the appropriate amino acids on the mRNA templates.

2. Primary structure of tRNAs

a. tRNAs are short, being only 73 to 93 nts long.

b. All tRNAs have the trinucleotide CCA at the 3' end.

(1) The amino acid is attached to the terminal A of the CCA.

(2) In most prokaryotic tRNA genes, the CCA is encoded at the 3' end of the gene. No known eukaryotic tRNA gene encodes the CCA, but rather it is added posttranscriptionally by the enzyme tRNA nucleotidyl transferase.

c. tRNAs have a large number of modified bases.

Over 50 different post‑transcriptional covalent modifications are known in tRNAs, such as dihydrouridine (D), in which the double bond between C4 and C5 is reduced, or pseudouridine (y), in which C5 is replaced with a N, providing another endocyclic amino group. The modified bases are especially prevalent in the loops.

Figure 3.5.2. Secondary structure of tRNA.

3. The secondary structure of tRNA is a cloverleaf

a. tRNAs have 4 arms with 3 loops (see Figure 3. 5.2. for yeast phenylalanine tRNA)

b. The amino acid acceptor arm is formed by complementary base‑pairing between the intial 7 nts of tRNA and a short segment near the 3' end. Again, the amino acid will be added to the terminal A.

c. The D arm ends in the D loop. It contains several dihydrouridines, which are abbreviated "D".

d. The anticodon arm ends in anticodon loop. The anticodon is located in the center of the loop. It will align 3' to 5' with the mRNA (reading 5' to 3').

e. The variable loop varies in size in different tRNAs. The difference in size between the 73 nt versus 93 nt tRNAs is found in the variable loop.

f. The TyC arm is named for this highly conserved motif found in the loop.

4. The tertiary structure of tRNA is a "fat L".

See Fig 3.5.3.

a. Some nucleotides in the D loop form base pairs with some nucleotides in the TyC loop. These and other interactions bring the cloverleaf (secondary structure) into an inverted L shape, with the "additional" base pairs found mainly at the junction of the inverted L.

b. In the 3‑D structure, two RNA double helices are at right angles. One of the double helices is the TyC stem in line with the amino acid acceptor stem. The other double helix has the D stem in line with the anticodon stem.

c. The result is that the two "business ends" of the tRNA are widely separated in space, at the two extremes of the tRNA. That is, the amino acid acceptor site is maximally separated from the anticodon (Figs. 3.5.3).

d. The rest of the molecule is a complex surface that must be recognized accurately by aminoacyl‑tRNA synthetases.

Fig. 3.5.3. 3-D structure of tRNA

A chime tutorial on tRNA structure is available from Dr. William McClure’s website at Carnegie-Mellon University:

http://info.bio.cmu.edu/Courses/BiochemMols/tRNA_Tour/tRNA_Tour.html

B. Attachment of amino acids to tRNA

1. Aminoacyl‑tRNA synthetases

a. Approximately 20 enzymes, one per amino acid.

b. Must recognize several cognate tRNAs, i.e. that accept the same amino acid but recognize a different codon in the mRNA (a consequence of the degeneracy in the genetic code).

c. Must not recognize the incorrect tRNA ‑ i.e. these enzymes require precise discrimination among tRNAs.

d. Two different classes of aminoacyl‑tRNA synthetases

The two classes of enzymes are distinguished by the structure of their tRNA‑binding regions. The different classes of enzyme approach and bind to different faces of the tRNA, but both must recognize the ends as well as any distinguishing features of the their cognate tRNAs.

Each class has about ten synthetases (for ten amino acids).

Fig. 3.5.4. 3-D structure of Glutaminyl-tRNA synthetase

The two classes of enzymes do not resemble each other much at all, in either sequence or 3-D structure, leading to the suggestion that they have evolved separately. If so, this would imply that an early form of life may have evolved using the ten amino acids handled by one class (or the other) of synthetase.

2. Mechanism

a. Aminoacyl-tRNA synthetase catalyzes a 2 step reaction. (Fig. 3.5.5)

First the amino acid is activated by adenylylation, i.e. a mixed anhydride intermediate is formed between the COO- of the amino acid and the a‑phosphoryl of ATP, with the liberation of pyrophosphate. The intermediate (activated amino acid) is an aminoacyl‑AMP..

In the second step, the amino acid is transferred to the 3' (or 2') OH of the ribose of the terminal A of tRNA, with liberation of AMP.

b. The product aminoacyl‑tRNA retains a high energy bond in an ester linkage.

(a) The equilibrium constant is about 1 for each of the two reactions, so the high energy of the bond initially between the a and b phosphoryls of ATP is essentially still present in the ester between the amino acid and the ribose of tRNA.

(b) The high energy bond in aminoacyl‑tRNA provides a driving force for protein synthesis.

c. Hydrolysis of pyrophosphate (abbreviated PPi) to two phosphates provides the free energy to drive synthesis of the aminoacyl-tRNA.

Thus one can consider that the equivalent of 2 ATPs (i.e. two high energy bonds) are used to form aminoacyl-tRNA, but one of the high energy bonds is retained in the product.

ATP ‑> AMP + PPi

PPi ‑> 2 Pi

In both instances, the cognate tRNA must be bound before proofreading can occur.

Figure 3.5.5.

3. Precise discrimination by AA‑tRNA synthetases

a. These enzymes must recognize the correct tRNA and the correct amino acid at the initial binding steps.

b. Proofreading is the removal of the incorrect amino acid (or tRNA) after binding, and often after part of the enzymatic reaction has occurred.

This can occur at either of the two reactions ‑ some synthetases will cleave an incorrect aminoacyl‑adenylate intermediate, and others will add the incorrect amino acid to the tRNA before recognizing the mistake and cleaving off the incorrect amino acid.

C. Anticodon determines specificity

The anticodon determines specificity for incorporation into a polypeptide during translation, not the amino acid. This was shown in the following experiment.

a. Cys‑tRNAcys can be converted to Ala‑tRNAcys by reductive desulfuration (H+ and Raney nickel), releasing H2S.

b. The resultant Ala‑tRNAcys retains the ACA anticodon to match a UGU codon in mRNA. When tested in cell‑free translation, it causes alanine to be incorporated instead of cysteine. (Fig. 3.4.4.)

c. Thus the amino acid on the tRNA did not direct its incorporation into the growing polypeptide chain, the anticodon did.

Figure 3.5.6.

D. Special tRNA for intiation of translation

1. Although Met has a single codon, two different tRNAs with different functions recognize the AUG codon.

(1) tRNAfmet (often abbreviated tRNAf) is used for initiation or translation in bacteria. A comparable initiator tRNA, called tRNAi , is used in eukaryotes.

(2) tRNAmmet is used for elongation.

Figure 3.5.5.

2. In bacteria, a formyl group is added to the amino group on the charged Met‑tRNAf , using 10‑formyl‑tetrahydrofolate as the formyl donor. This prevents its use in elongation.

3. In bacteria, only formylmethionyl‑tRNAf can bind to the partial P site on the small ribosomal subunit (see below) to initiate translation at AUG, or GUG (less frequently) or UUG (rarely). In all three cases, the protein starts with formylmethionine. The formyl group is removed after the first several amino acids have been incorporated, and in about half the cases, the methionine is also removed.

4. Note that the meaning of AUG and GUG is dependent on the context. AUG or GUG at the initiation site encodes formyl‑Met, but when internal to the mRNA, they encode Met or Val, respectively.

5. tRNAf has a different structure from tRNAm, and these differences determine their use either in initiation or elongation.

6. In eukaryotes, Met‑tRNAi is used for initiation. Although it is not formylated, the basic process is similar to that in prokaryotes.

E. Ribosomes

1. Role of ribosomes

a. Ribosomes are the molecular machines that catalyze peptide bond formation between a growing polypeptide and an incoming aminoacyl‑tRNA. The ribosomes insures that the amino acids are added in the order specified by the mRNA.

b. Ribosomes associate reversibly with the mRNA.

The two subunits of the ribosome form a complex around the mRNA to translate, and then dissociate after translation is completed.

2. Size and Composition of large and small subunits (see Fig.3.5.6.).

a. Ribosomes ("ribonucleic acid" "bodies") are large complexes of RNA and protein, with a roughly 60:40 ratio between RNA and protein. There are two subunits. Similar components are found in both eukaryotes and prokaryotes, although their sizes differ.

b. Each subunit has one major RNA (in bacteria, 23S rRNA for the large subunit and 16S for the small subunit) and many proteins (31 and 21, respectively, for bacterial large and small subunits). The large subunit also has a small rRNAs about 120 nucleotides in size (5S RNA). Eukaryotic large ribosomal subunits have an additional small RNA (5.8S) that corresponds to the sequence of the 5' end of bacterial 23S rRNA.

The bacterial ribosome is composed of three different RNA molecules and more than 50 different proteins arranged in two major subunits, which join together to form the complete ribosome. During protein synthesis, the ribosome binds transfer RNA molecules in three different sites. In this image of the ribosome with transfer RNAs in all three binding sites, the large subunit is gray, the small subunit is violet, and the three transfer RNAs are green, blue, and red. Image is from the Center for Molecular Biology of RNA, http://currents.ucsc.edu/99-00/09-27/ribosome.art.html

Figure 3.5.8. Images of ribosomes based on 3-D structure determination. The top view is from the Noller lab at UCSC, the bottom is from the Steitz lab and collaborators at Yale. The bottom view shows the RNA in silver ribbons and protein as gold coils. A green tRNA is at the peptidyl transferase site. Image from http://www.npaci.edu/features/01/05/05_03_01.html

c. The rRNAs and subunits were initially characterized by their sedimentation velocity, and hence are referred to by their sedimentation value in Svedberg units, or S. Larger macromolecules and complexes sediment faster and have a higher S value. However, other factors play a role in sedimentation rate (such as shape) and the S values for a complex is not the sum of the S values of individual components.

3. Shape

a. The small subunit is fairly elongated and binds mRNA.

b. The large subunit is more spherical and covers the small subunit.

c. The mRNA may thread between the 2 subunits or it may lie outside the ribosome.

4. P (peptidyl‑tRNA) and A (aminoacyl‑tRNA) and E (exit) sites

A tRNA interacts with the ribosome at three major sites as it brings in an amino acid, has the growing polypeptide chain attached to that amino acid, and then finally leaves the ribosome after donating its amino acid.

a. A site (or entry site): aminoacyl‑ tRNA binds

b. P site (or donor site): peptidyl‑tRNA binds, i.e. the nascent polypeptide chain linked to the last tRNA to occupy the A site (see below).

c. E site: exit of deacylated tRNA after peptide bond formation.

d. Flow of tRNA through the ribsoome is from the A site to P site, then exit via the E site.

e. The next point will become clearer after we discuss the elongation cycle. The molecule attached to the 3' end of the tRNA is different at each site.

Fig. 3.5.9.

F. The polarity of translation is from the amino (N) terminus to the caboxy (C) terminus.

This was demonstrated in a classic experiment by Dintzis.

1. Actively translating proteins were labeled with radioactive amino acids for a brief time (short relative to the time required to complete synthesis).

2. Completed polypeptides were collected, digested with trypsin, and the amount of radioactivity in tryptic fragments was determined.

3. Tryptic fragments from the C‑terminal end of the polypeptide had radioactivity at the earliest times of labeling.

4. As the period of labeling was increased (longer pulse), tryptic fragments closer to the N terminus were labeled.

5. This shows that the direction of polypeptide growth is from the N teminus to the C terminus, i.e. translation begins at the N terminal amino acid. This corresponds to mRNA chain growth in a 5' to 3' direction.

6. Note that this experimental protocol is also used to map origins of replication, as we covered in Part Two of the course.

Fig. 3.5.10.

G. Initiation of translation

1. mRNA binds to small ribosomal subunit (not the whole 50S ribosome) in such a way that the initiator AUG is positioned in the precursor to the P site, i.e. ready for the f-met-tRNAfmet to recognize it.

a. The alignment of the initiator AUG in the mRNA with the appropriate place on the ribosomal subunit involves base pairing between the 3' end of 16S rRNA and a sequence that precedes the initiator AUG in mRNA. When this portion of 16S rRNA in the 23S subunit is removed by cleavage with colicin (an antibiotic), the 23S subunit loses the ability to initiate translation.

Figure 3.5.11.

b. The ribosome binding site is in the 5' untranslated region, just before the initiator AUG. It is also called a Shine‑Dalgarno sequence (named for the discoverers of the sequence).

It is a purine‑rich sequence, e.g. 5' AGGAG, that will pair with the pyrimidine‑rich 3' end of 16S rRNA (5' CCUCCUUA‑OH 3')

c. This base pairing insures the choice of the correct AUG as initiation codon, as opposed to an internal AUG.

2. Roles of initiation factors and other factors

a. Translation factors are used at only one step of the process and are not permanent subunits of the ribosome. They cycle on and off the ribosomes as they do their function. They are (frequently) present in smaller amounts than the ribosomal subunits.

Figure 3.5.12.

b. IF3 = Initiation Factor 3

(1) An antiassociation factor; prevents association between the large and small ribosomal subunits.

(2) It also must be associated with the small subunit for it to form an initiation complex, i.e. for the small subunit to correctly bind mRNA and fmet-tRNAf.

(3) It dissociates prior to binding of the large subunit.