As was discussed in Chapter 2 , the messenger RNA (mRNA) is transcribed from the DNA coding sequence by RNA polymerase using nucleic acids, creating an antiparallel RNA strand. This RNA compliment contains the same nucleic acids as DNA, with the exception of thymine, which is replaced in RNA by a nucleic base called uracil.
The structure of DNA was discovered by Watson and Crick among others, such as the contribution of Rosalind Franklin, whose X-ray crystallography of the DNA molecule was the instrumental confi rmation. After this important discovery, many scientists concentrated on the transfer of the information contained in the DNA molecule to the RNA molecule and eventually to de nova protein production within the cell. It was Crick and Brenner et al. that fi rst demonstrated that the codon usage consisted of three DNA bases. In 1961, Marshall Nirenberg and Heinrich Matthaei demonstrated that three base sequences, such as UUU, were the codons that translated
the information from the RNA to the protein amino acid phenylalaine. Through a series of similar experiments by other groups, such as Severo Ochoa, the rest of the codon sequences were determined ( Figure 4.1 ). Because of this ground- breaking work on RNA synthesis, Ochoa received the Nobel Prize in Physiology or Medicine in 1959. Ultimately it was Nirenberg and Leder who solved the triplet organization of the codon and thus the common genetic code for all species [85]. For this work Nirenberg, among others, received the Nobel Prize in Physiology or Medicine in 1968. Among these was Robert W. Holley for his work in determining the structure of the transfer RNA (tRNA), the adapter molecule that transfers the correct amino acid to the C-terminal end of the growing protein sequence.
There can be as many as six codons used for one amino acid, such as arginine (R), luecine (L) and serine (S).
Conversely, there are only two amino acids, methionine (M) and tryptophan (W), which have only one codon ( Table 4.1 ).
Prokaryotic ribosomal composition Figure 4.1
Codons for amino acids and start and stop sequences
Table 4.1
Amino acid
Codons Compressed Amino acid
Codons Compressed
Ala/A GCU, GCC, GCA, GCG
GCN Leu/L UUA, UUG, CUU, CUC, CUA, CUG
YUR, CUN
Arg/R CGU, CGC, CGA, CGG, AGA, AGG
CGN, MGR Lys/K AAA, AAG AAR
Asn/N AAU, AAC AAY Met/M AUG
Asp/D GAU, GAC GAY Phe/F UUU, UUC UUY
Cys/C UGU, UGC UGY Pro/P CCU, CCC, CCA, CCG
CCN
Gln/Q CAA, CAG CAR Ser/S UCU, UCC, UCA, UCG, AGU, AGC
UCN, AGY
Glu/E GAA, GAG GAR Thr/T ACU, ACC, ACA, ACG
ACN
Gly/G GGU, GGC, GGA, GGG
GGN Trp/W UGG
His/H CAU, CAC CAY Tyr/Y UAU, UAC UAY
Ile/I AUU, AUC, AUA
AUH Val/V GUU, GUC, GUA, GUG
GUN
START AUG STOP UAA, UGA,
UAG
UAR, URA
Note: Inverse table (compressed using IUPAC notation)
Once the mRNA strand is made, it is then able to be translated by the ribosomal complex into a protein sequence, which is folded into its correct native conformation. The ribosomal complex is made up of the two ribosomal subunits, 50S and 30S. It was in the 1950s that ribosomes were fi rst observed by cell biologist George Emil Palade using an electron microscope. It cannot be overstated how important is the continued understanding of the ribosomal complex in terms of its action and structure within the cell.
In both prokaryotic and eukaryotic cells, the ribosomal complexes consist of two subunits that fi t together ( Figure 4.1
Translation of protein in prokaryotes (Wikipedia) Figure 4.2
represents the prokaryotic ribosomal complex) and work as one to translate the mRNA into a polypeptide chain during protein synthesis ( Figure 4.2 ). Prokaryotic ribosomes are around 20 nm (200 Å) in diameter and are composed of 65%
ribosomal RNA (rRNA) and 35% ribosomal proteins.
Eukaryotic ribosomes are between 25 and 30 nm (250 to 300 Å) in diameter. Bacterial subunits generally consist of two subunits, while eukaryotic subunits consider of one or three very large RNA molecules (known as ribosomal RNA or rRNA) and multiple smaller protein molecules.
Crystallographic work has shown that there are no ribosomal proteins close to the reaction site for polypeptide synthesis.
This proves that the protein components of ribosomes do not directly participate in peptide bond formation catalysis, but rather suggests that these proteins act as a scaffold that may enhance the ability of rRNA to synthesize protein. The ribosomal subunits of prokaryotes and eukaryotes are similar [86,87]. The unit of measurement of the ribosomal complex
and subunits is the Svedberg unit, a measure of the rate of sedimentation in centrifugation rather than size, and this accounts for why fragment names do not add up (70S is made of 50S and 30S).
As shown in Figure 4.1 , prokaryotes have 70S ribosomes, each consisting of a small (30S) and a large (50S) subunit.
Their small subunit (30S) has a 16S RNA subunit, consisting of 1540 nucleotides and 21 proteins bound together. The large subunit (50S) is composed of RNA subunits 5S, containing 120 nucleotides and 23S RNA containing 2900 nucleotides. Also contained in the 50S ribosomal subunit are 31 proteins [87]. The affi nity label for the tRNA binding sites on the E. coli ribosome allowed for the identifi cation of A and P site proteins most likely associated with the peptidyltransferase activity, by Collatz and Czernilofsky [88,89]. Additional research has demonstrated that the S1 and S21 proteins, in association with the 3ʹ-end of 16S rRNA, are involved in the initiation of translation [89].
Like prokaryotes, eukaryotes have a complex of small and large ribosomal subunits totaling a Svedberg number of 80S.
The small subunit (40S) contains an 18S RNA of 1900 nucleotides bound together with 33 proteins [90,91]. The large subunit (60S) consists of three RNA subunits, the 5S of 120 nucleotides, the 28S of 4700 nucleotides and a 5.8S subunit of 160 nucleotides. These three RNA subunits are also associated with 46 different proteins [92]. During 1977, Czernilofsky published research that used affi nity labeling to identify tRNA-binding sites on rat liver ribosomes. Several protein complexes, including L32/33, have been implicated as being at or near the peptidyl transferase center [93].
Interestingly enough, ribosomes found in chloroplasts and mitochondria of eukaryotes also consist of large and small subunits bound together with proteins into one 70S particle [86]. These organelles are believed to be descendants of
bacteria and as such their ribosomes are similar to those of bacteria [94].
The various ribosomes share a core structure, which is similar despite the large differences in size. Interestingly, the greater percentage of RNA molecules are highly organized into various tertiary structural motifs. These structures, for reasons not clearly understood, are organized into knot- like, stacked arrangements with a common central axis. Within the larger ribosomal subunit, the RNA is found in large continuous loops that come out of the core of this structure without disrupting or changing it [95]. Furthermore, the proteins in the ribosome exhibit no catalytic activity and seem to be only structurally required by stabilizing the ribosomal structure. All of the catalytic activity of the ribosome is carried out by the RNA [96].
For all their similarities, there are important differences between the bacterial and eukaryotic ribosomes. These differences are taken advantage of by pharmaceutical chemists to create antibiotics that can destroy a bacterial infection without harming the cells of the infected person.
Due to the differences in their structures, the bacterial 70S ribosomes are susceptible to these antibiotics, while the eukaryotic 80S ribosomes are not [97]. However, what about the mitochondrial ribosomes? Even though they are very similar to the bacteria, they are not affected by these antibiotics because they are surrounded by a double membrane that acts as a suffi cient barrier, preventing introduction of the antibiotics into the organelle [98].
4.2.1 Initiation of translation
These two subunits combine together with the mRNA, the fi rst aminoacyl tRNA, which carries the fi rst amino acid
methionine (RNA code AUG), and three initiation factors (IF1–3). The reaction is stimulated by GTP, which is carried on the IF2 factor. The ribosomal complex is fi rst formed by the combination of IF1, IF2 and IF3 with the small ribosomal subunit 30S. Once this initiation complex is formed it binds to the mRNA in a region 6–7 bases upstream from the start codon AUG. This binding site, called the Shine-Dalgarno sequence or box (AGGAGG) in prokaryotes, fi rst discovered by John Shine and Lynn Dalgarno, allows the initiation complex to bind next to the start codon, forming a double stranded RNA structure. The tRNA, containing a coding region of three bases, is the anticodon of the mRNA sequence for methionine. To enable the translation of the mRNA into a protein sequence, there is a tRNA that is specifi c for each amino acid. This tRNA, while carrying an amino acid, also carries the anticodon sequence for a three- base code representing the amino acid.
4.2.2 Elongation of translation
Once the ribosomal complex has settled on the starting codon AUG, the elongation phase of translation begins.
Elongation is the process where amino acids are consecutively added onto the growing end of the C-terminal end of the protein. Not shown in the Figure 4.2 , but present in the 50S ribosomal subunit, are the P and A sites. During the fi rst step of the elongation, the fMet- tRNA enters the P site, causing a structural rearrangement of the 50S subunit to reveal the A site, which allows for a new aminoacyl tRNA to bind to the mRNA. This mechanism is facilitated by the elongation factor EF-Tu, which is an enzyme called a GTPase.
Once the P and A sites are fi lled, the fi rst tRNA is released from the methionine amino acid and a catalytic reaction
A tetrapeptide (V-G-S-A) with the amino terminus of the peptide on the left and the carboxyl terminus on the right. Each of the peptide bonds was made by combining a free N-terminus and C-terminus in a dehydration reaction that releases H 2 O
Figure 4.3
takes place between the methionine amino acid and the next amino acid in the A site, forming a peptide bond between the two molecules ( Figure 4.3 ). This peptide bond is catalyzed by the 23S rRNA residing in the 50S subunit. Now the P site contains an uncharged tRNA, which moves to the E site, and the A site contains a dipeptide molecule called a dipeptidyl- tRNA, which now moves into the open P site leaving the A site ready to receive the next aminoacyl- tRNA with the help of EF-Tu. The movement of deacylated tRNA and the dipeptidyl- tRNA is coordinated by the elongation factor G (EF-G), allowing the deacylated tRNA to be released from the E site. This process occurs over and over again until the ribosomal complex reaches a stop codon. Interestingly, the prokaryotic translation machinery is relatively slow compared to DNA replication. Only 18 proteins per second are made on average. This is most likely due to the mis- reading of the mRNA and the time it takes to reject the incorrect amino acid and wait for the correct one to come along.
4.2.3 Termination of translation
Reaching the termination codons or stop codons (UAA and UAG), the tRNA will not recognize either of these codons but a release factor such as RF1 or RF2. These release factors catalyze the hydrolysis of the ester bond between peptidyl- tRNA in the last amino acid of the sequence. Then a third release factor (RF3) presents itself and catalyzes the release of the RF1 and RF2 factors. The ribosomal complex is then released from the mRNA by the Ribosome Recycling Factor and EF-G, which also helps in its disassociation into individual components, to then be used for another round of translation [99].