Protein Synthesis And DNA Mismatch Repair: Molecular Mechanisms And Pathways

Protein Synthesis: Initiation, Elongation, Termination, and Recycling Steps

1. The process of protein synthesis from messenger RNAs or mRNAs is called translation. The 4 phases of protein synthesis are initiation, elongation, termination and ribosome recycling (Hershey, Sonenberg and Mathews 2012). In order to start protein synthesis, the mRNAs must be recruited onto a ribosome. Translation initiation is highly regulated step of the protein synthesis pathway. The initiation of protein synthesis has 5 steps, which are: binding of mRNA by the eIF4F cap binding complex, formation of 43S pre-initiation complex, recruitment of mRNA to the ribosome, localization of the initiation codon and joining of the 60S ribosome (Hinnebusch and Lorsch 2012). Selection of mRNAs for translation depends on the presence of 2 features. These are the 5’7-methyl guanosine cap and the 3’ poly A tail (Furuichi 2015). The function of the eukaryotic translation initiation factor 4F or eIF4F is to binds the 5’ end of the mRNA with the help of the 5’7-methyl guanosine cap. The eIF4F ensures the binding of the 40S ribosome on the 5’ end of the mRNA by removing  any inhibitory secondary structures. The eIF4F consists of 3 subunits. These are the eIF4E, eIF4A and eIF4G (Merrick 2015). The recognition of the 5’7-methyl guanosine cap by the eIF4E is essential for mRNA recruitment. eIF4E also interacts with eIF4G and positions it on to the 5’ end of the mRNA. This helps to ensure the attachment of the mRNA on the 40S ribosome. The function of the eIF4G is to stabilize the interaction of eIF4E with the cap region and also helps to stabilize the interactions of the poly A binding protein or PABP with the poly A tail (Goss and Kleiman 2013). eIF3 remains associated with the 40S ribosomal subunit and prevents the premature binding of the 60S ribosomal subunit. eIF3 interacts with the eIF4F complex. eIF4A is a ATP dependent RNA helicase that helps the ribosome to resolve secondary structures present in the mRNA transcript. The 43S pre-initiation complex consists of the 40S small ribosomal subunit, initiation factors eIF1, eIF1A, eIF3 and the eIF2-Met-tRNA^Met-GTP ternary complex (Hussain et al. 2014). The 43S pre-initiation complex along with the other protein factors scans the mRNA chain towards its 3’ end in order to reach the start codon. This process is called scanning (Lee et al. 2012). The start codon encodes the amino acid methionine. The eIF2 brings the methionine charged initiator tRNA to the P site of the 40S ribosomal subunit. It catalyzes the hydrolysis of GTP and brings about the disassociation of the initiation factors thereby enabling the binding of the large ribosomal subunit called the 60S subunit. The complete 80S ribosome thus formed helps to initiate the translation elongation steps. After initiation, in the elongation step the mRNA is positioned in order to enable the translation of the next codon. The initiator tRNA binds to the P site of the ribosome, while the A site of the ribosome remains free for binding to the next aminoacyl tRNA, which is specific for the desired codon. This step of the elongation phase is followed by the formation of peptide bonds and shifting of the mRNA by one codon with respect to the ribosome. Thus, the shifting ensures that a new codon occupies the A site of the ribosome. The 2 eukaryotic elongation factors are the eEF-1 and eEF-2 (Sasikumar, Perez and Kinzy 2012). eEF-1 ensures the entry and binding of the incoming aminoacyl tRNA to the free site of the ribosome that is the A site. It has 2 subunits. These are α and βγ. The α subunit is responsible for mediating the binding of the aminoacyl tRNA to the A site and the βγ subunit functions as a guanine nucleotide exchange factor thereby helping to release the GDP from the α subunit. The eEF-2 elongation factor is responsible for mediating the translocation of the mRNA and the aminoacyl tRNA along the ribosome after each peptide bond formation. Peptide bond formation is mediated by the peptidyl transferase enzyme and this enzyme activity lies on the 28S rRNA of the 60S ribosomal subunit. Formation of peptide bond is followed by the translocation of the growing peptide chain to the P site from the A site. Translocation is carried out by eEF-2 and GTP (Lareau et al. 2014). The termination step of translation depends on the universal release factor eRF-1. This release factor recognizes all the three stop codons and stimulates the events associated with the termination step. When the release factor binds to the stop codon, the ribosome subunits disassemble and fall off. The completed polypeptide chain is also released. eRF3 is ribosome dependent GTPase that helps in the release of the complete polypeptide chain (Preis et al. 2014). Prokaryotic translation also involves the 4 steps of initiation, elongation, termination and recycling. The initiation involves the 50S and 30S ribosomal subunits, mature mRNA, N- formylmethionine and GTP. The prokaryotic initiation factors are IF1, 2 and 3. The 30S ribosomal subunit binds to the Shine Dalgarno sequence of the mRNA. This places the initiation codon at the P site. In the elongation step, the formyl methionyltRNA enters the P site and the A site remains free to bind to a new incoming aminoacyl tRNA. EF-Tu is a GTPase that facilitates the binding. The ribozyme named 23S rRNA are involved in the catalysis of peptide bond formation. The dipeptidyl tRNA in the A site then undergoes translocation to the P site and the deacylated tRNA at the P site moves to the E site. This process is facilitated by EF-G. When the ribosome reaches the termination codon, release factors called RF-1, 2 and 3 participate in the process. RF-1 recognizes the stop codons UAA and UAG, while RF-2 recognizes UAA and UGA. They catalyze the release of the newly synthesized polypeptide chain. RF-3 catalyzes the release of RF-1 and 2. The ribosome recycling factor functions to release the mRNA and the deacylated tRNAs from the ribosomes. The ribosome also undergoes disassociation to 50S and 30S (Prabhakar, Chen and Puglisi 2016).

Components and Functions of Eukaryotic and Prokaryotic Translation Factors

                               

                                               Figure 1: Scanning mechanism in eukaryotic translation

                                                                (Source: Poulin, F. and Sonenberg 2013)

2. DNA mismatch repair recognizes the errors occur during DNA replication, recombination and also repairs various forms of damage to the DNA. It recognizes and repairs erroneous insertions, deletions or mis-incorporation of bases. Such errors occur during DNA replication and recombination. Mismatch repair systems functions to maintain the genome stability during repeated rounds of replication. The essential components of the prokaryotic mismatch repair system are the MutL, MutH, MutS and Uvr. MutS helps to detect mismatches in the DNA and initiates the mismatch repair mechanism (Elez, Radman and Matic 2012). The MutS recognizes a specific region of mismatch and undergoes a conformational change that helps to unbend the DNA. MutS homodimers bind to heteroduplex DNA. MutS has 2 domains. These are the DNA binding domain and the ATPase dimerization domain. A glutamate residue of MutS helps to form a hydrogen bond with the mismatch base and a conserved phenylalanine residue of MutS is also involved in the binding to the mismatch base. After this interaction, MutS initiates the mismatch repair system by directly or indirectly interacting with other proteins. These proteins are MutL, MutH and UvrD. The function of MutL is to recognize the mismatch and enable excision of the mismatch from the DNA strand. A homodimer of MutL brings about its interaction with MutS and in turn activates the endonuclease activity of MutH. MutL helps in the loading of the UvrD onto the DNA. The DNA helicase activity of UvrD helps to unwind the DNA at the region of the nick produced by MutH. MutH is a type II restriction endonuclease that cleaves at the hemimethylated GATC sites, thereby bringing about an excision of the mismatch containing DNA strands. The MutH endonuclease activity is mediated by MutS, MutL and ATP. The nick formed in the DNA strand by MutH allows the entry of UvrD/helicase II and single strand DNA binding proteins. These proteins are loaded onto the nick by interactions with MutL. The newly synthesized strand is excised between the nick and the mismatch. This process is carried out by the single strand DNA specific exonucleases, which are designated ExoI, ExoX, RecJ and ExoVII.  ExoI and ExoX have 3’ – 5’ exonuclease activity and the RecJ and ExoVII have 5’ -3’ exonuclease activity. The repair synthesis is carried out by the DNA polymerase III, DNA ligase and single strand DNA binding protein (Erie and Weninger 2014). The eukaryotic mismatch repair system consists of the MutS and MutL homologs. These are called MSHs and MLHs, respectively. Although MutS and MutL form homodimers. The MSHs and MLHs form heterodimers with a variety of proteins. There are 5 highly conserved MSHs. These are MSH2, 3, 4, 5 and 6. MSH2 is required for mismatch repair of nuclear DNA, while MSH3 and MSH6 are involved in the repair of DNA mismatches that are highly distinct and overlapping and such mismatches have been found to occur during replication. The MutSα homologs consisting of MSH2 and 6 plays an essential role in the recognition of mismatched DNA, while the MutSβ homolog consisting of MSH2 and 3 plays an essential role in repairs of mispairs in insertion or deletion loops. The  MutSα also functions in the repair of mispairs in insertion or deletion loops and base to base mispairs. The MutL homologs in eukaryotes have been termed Mlh 1and Pms 1. They form heterodimers with each other. The eukaryotic MutL has three forms. These are MutLα, β and γ. The MutLα consists of 2 subunits namely MLH1 and PMS2. The MutLβ consists of MLH1 and PMS1 and the MutLγ consists of MLH1 and MLH3. MutLα helps in coordinating the mismatch repair events and also functions as an endonuclease that introduces breaks in the DNA strand upon activation by the mismatch and other proteins like the PCNA and the MutSα. PCNA or the proliferating cell nuclear antigen increase or enhances the binding of MutSα to the mismatched regions of the DNA. It also enables MutSα to search for the presence of mismatches in the DNA. Eukaryotes also use exonuclease activities of ExoI, which is a 5’ – 3’ exonuclease, which plays a role in mutation avoidance as well as mismatch repair in yeasts. The mammalian ExoI is also involved in both 5’ and 3’ nick directed excisions. The replication protein A or RPA is a single strand DNA binding protein and involved in excision. In the presence of PCNA and the replication factor C, 3’ – 5’ excision is carried out by the MutSα, MutLα, ExoI and RPA. Once strand excision occurs, polymerase carries out DNA re-synthesis with the help of PCNA and RPA. The resulting nick is sealed by a DNA ligase (Kunkel and Erie 2015).

                   

                                                                           Figure 2: Mismatch repair

                                                                     (Source: Hsieh and Zhang  2017)

Reference List

Elez, M., Radman, M. and Matic, I., 2012. Stoichiometry of MutS and MutL at unrepaired mismatches in vivo suggests a mechanism of repair. Nucleic acids research, 40(9), pp.3929-3938.

Erie, D.A. and Weninger, K.R., 2014. Single molecule studies of DNA mismatch repair. DNA repair, 20, pp.71-81.

Furuichi, Y., 2015. Discovery of m7G-cap in eukaryotic mRNAs. Proceedings of the Japan Academy, Series B, 91(8), pp.394-409.

Goss, D.J. and Kleiman, F.E., 2013. Poly (A) binding proteins: are they all created equal?. Wiley Interdisciplinary Reviews: RNA, 4(2), pp.167-179.

Hershey, J.W., Sonenberg, N. and Mathews, M.B., 2012. Principles of translational control: an overview. Cold Spring Harbor perspectives in biology, 4(12), p.a011528.

Hinnebusch, A.G. and Lorsch, J.R., 2012. The mechanism of eukaryotic translation initiation: new insights and challenges. Cold Spring Harbor perspectives in biology, 4(10), p.a011544.

Hsieh, P. and Zhang, Y., 2017. The Devil is in the details for DNA mismatch repair. Proceedings of the National Academy of Sciences, 114(14), pp.3552-3554.

Hussain, T., Llácer, J.L., Fernández, I.S., Munoz, A., Martin-Marcos, P., Savva, C.G., Lorsch, J.R., Hinnebusch, A.G. and Ramakrishnan, V., 2014. Structural changes enable start codon recognition by the eukaryotic translation initiation complex. Cell, 159(3), pp.597-607.

Kunkel, T.A. and Erie, D.A., 2015. Eukaryotic mismatch repair in relation to DNA replication. Annual review of genetics, 49, pp.291-313.

Lareau, L.F., Hite, D.H., Hogan, G.J. and Brown, P.O., 2014. Distinct stages of the translation elongation cycle revealed by sequencing ribosome-protected mRNA fragments. Elife, 3, p.e01257.

Lee, S., Liu, B., Lee, S., Huang, S.X., Shen, B. and Qian, S.B., 2012. Global mapping of translation initiation sites in mammalian cells at single-nucleotide resolution. Proceedings of the National Academy of Sciences, 109(37), pp.E2424-E2432.

Merrick, W.C., 2015. eIF4F: a retrospective. Journal of Biological Chemistry, 290(40), pp.24091-24099.

Poulin, F. and Sonenberg, N., 2013. Mechanism of translation initiation in eukaryotes.

Prabhakar, A., Chen, J. and Puglisi, J.D., 2016. The Dynamic Pathways of Prokaryotic Translation Termination and Recycling. Biophysical Journal, 110(3), pp.351a-352a.

Preis, A., Heuer, A., Barrio-Garcia, C., Hauser, A., Eyler, D.E., Berninghausen, O., Green, R., Becker, T. and Beckmann, R., 2014. Cryoelectron microscopic structures of eukaryotic translation termination complexes containing eRF1-eRF3 or eRF1-ABCE1. Cell reports, 8(1), pp.59-65.

Sasikumar, A.N., Perez, W.B. and Kinzy, T.G., 2012. The many roles of the eukaryotic elongation factor 1 complex. Wiley Interdisciplinary Reviews: RNA, 3(4), pp.543-555.