User talk:Nghaemim



DNA is replicated through the interaction of the template strand with a huge protein complex called the replication complex, which catalyze the reaction involved. All chromosomes have at least one base sequence, called the origin of replication (ori), to which this reapplication complex initially binds. This binding is based on the recognition of the different nucleotide bases by proteins. DNA replicates in both directions from the origin of replication, forming two replication forks. Both of the separated strands of the parent molecule act as templates simultaneously, and the formation of the new strands is guided by complementary base pairing. Replication begins at specific “origin of replication” sequences to which this replication complex initially binds, and forms replication “bubbles” Two important rules for DNA synthesis are DNA polymerases cannot initiate a strand, and DNA synthesis always proceeds in the 5’ to 3’ direction. DNA needs a complementary short single strand of RNA which is called primer in addition to template in order to be initiated. For the second rule, when nucleotide are added to free hydroxyls, not to 5’ phosphate, we see that the template is actually traversed in the 3’ to 5’ direction, since the two strands are antiparallel. Thus, how can the “lagging” strand be replicated? The answer is discontinuously in other words, by way of Okazaki fragments. In E. coli at least, both strands are synthesized by a single enzyme complex. All replication complexes contain several proteins with different roles in DNA replication. Some of many proteins required for DNA replication in E-coli are being considered as below. An origin binding protein which is called DnaA initiates the process of replication by recognizing and binding to oriC, partially unwinding the helix. A helicase (DnaB protein) is there to continue the unwinding process. Complex needs another protein for releifing the tension and converted to happy way that was before by cutting both of the sugar phosphate back bone and move one of the ends around and then relegate. This protein is called a topoisomerase for “relaxing” the overwound DNA that accumulates ahead of helicase. There is another enzyme (primase) which is an RNA polymerase that lays down short about15 nucleotides lengths of RNA, priming DNA synthesis. Other protein is single stranded binding protein, ssb. Its function is to multiple copies of this relatively simple protein adhere to any single stranded DNA, preventing it from reattaching. Another important protein in the replication complex is a DNA polymerase III “dimmer” (1 monomer for each strand). In E. coli, DNA polymerase III is the primary replicative enzyme. Each monomer is very large, about 600 kilodaltons, complex of 10 subunit polypeptides. The alpha subunit contains the active site for nucleotide addition, and a dimmer of the beta subunit forms a doughnut-shaped ring surrounding one strand of DNA, preventing the polymerase from falling off. This called beta clamp. The epsilon subunit is responsible for proofreading. DNA polymerase I is responsible for removing the RNA primers and replacing them, one nucleotide at a time, with deoxyribonucleotide (DNA). It also functions in various DNA repair mechanisms. Finally, the enzyme DNA ligase is there for joining the Okazaki fragment after DNA polymerase I has acted. There is a model to show DNA replication process. Accuracy and speed in E. coli is about 500 nucleotides strand per second. The error rate of the polymerase III alpha subunit is 1/10000. This would be intolerably high. The proofreading activity of the epsilon subunit corrects most of these errors. A variety of other DNA repair system correct all manner of environmentally-induced mutations, and one of these the MutHLS system in E. coli, also corrects mismatched nucleotides in freshly synthesized DNA. The final correct rates is 1/1000000000 (1 mistake per billion nucleotides added). Numerous systems exist for repairing almost all imaginable types of mutation. In E. coli, over 100 genes are devoted to this protective function.

References: DNA replicases from a bacterial prespective,McHenry CS. Department of chemistry and biochemistry. University of Colorado,Boulder,Colorado 80309.USA. charles.McHenry@Colorado.edu

Assembly of Bacterial Ribosomes
The ribosome is a ribonucleoprotein complex responsible for one of the key processes in cells—protein synthesis. The protein sediments during ultracentrifugation as a 70S particle composed of a small subunit (30S) and a large subunit (50S).

Ribonucleoprotein complex is a ribosome, and its function is to synthesis of protein. When the protein will be ultra-centrifuged, the sedimentation shows that ribosome is consisting of 70S particle composed of a small subunit (30S) and a large subunit (50S).

During translationinitiation, the 30S subunit bonds with mRNA, and makes it ready mRNA decoding. Then, the 50S subunit comes and binds to mRNA and small subunit. It is the site for tRNA to come and bind to mRNA. Now, the 3-nt codon of tRNA is responsible to determine which amino acid to incorporate into the polypeptide chain next.

30S subunit contains pre-16S. In contrast, 50S subunit contains pre-23S. The5’ and 3’ endsin complete 30S subunits are far apart from each other, whereas the 5’ and 3’ ends in 50S are base-paired.Consequently, the maturation from pre-16S to complete16S within 30S particle activates the 30S subunits.

Tissieres & Watson proved that the bacterial ribosome is constituted of a 70S unit that is able to be broken down into two smaller parts, the small and large subunit, and that the 100S unit was a combined of the 70S.

In Vitro Assembly Map and Assembly Intermediates

In the late 1960s, a momentous happened, when Trauband & Nomura showed that an active 30S subunit may possibly be brought together in vitro from free rRNA and ribosomal proteins without any otherfactors. These in vitro re-formationtests proved that all of the information essential for getting-together is encoded within the rRNA and proteins themselves. Nomura and coworkers concluded that in vitro, protein-binding actions are thermodynamically inter-reliant, and that the ribosomal proteins bind to 16S rRNA in an ordered way. They proved that by changing the order in which the proteins were added. Primary binding proteins such as S4, S7, S8, S15, S17, S20 firmly bind directly to the new 16S rRNA. The secondary and tertiary binding proteins need previous binding of one or more proteins, correspondingly. At different temperatures (20, 47, 51, 52), for assembly of the 30S subunit, two intermediates were recognized by reconstitution. At 0°C to 15°C which is a low temperature, assembly stalls, and the reconstitution intermediate is formed. The reconstitution intermediate is made up of 16S rRNA and 15 ribosomal proteins, and it sediments at 21S-22S. To reconstitute again reconstitution intermediate should beheated to 40°C. At this point, RI changed to RI*. This is the second intermediate. The particle RI* sediments at 25S-26S. This RI* particle forms an active 30S subunit. To reconstitute active 50S subunits, Nierhaus & Dohme identified three reconstitution intermediates. These intermediates depend in the temperature and ionic conditions of the reconstitution. The first reconstitution intermediate RI50 (1) sediments at 33S. By heating of RI50 (1) at high temperature, it transits to the second intermediate RI50*(1) that sediments at 41S–43S.When the remaining proteins is added to the second intermediate, the third intermediate is formed. However, it is inactive and sediments at 48S. Heat should be increased and high magnesium ion concentrations should be present in order to form active 50S subunits.

The important Ribosomal proteins to transition of RI50 (1) to RI50*(1) are L4, L13, L20, L22, and L24 and 23S rRNA. At the 5′ of the 23S rRNA transcript, the essential ribosomal proteins bunch together.The ribosomal proteins L20 and L24 are important in the early stepsof assembly, but in the late steps their functions are not essential.

In Vivo Assembly Intermediates

Under standard growth situations, ribosomal assembly intermediates are not plentiful. Thus, early learning depends on “conditional mutants, temperature-sensitive strains, or fragile growth mutants to perturb growth and accumulate large amounts of potential ribosomal precursors, which are detectable on sucrose gradients” (Mangiarotti G 61-65). However, under standard growth conditions, incomplete ribosomal particles use pulse-labeling which allow really small quantities to be identified. Therefore, pulse labeling and polyacrylamide gels show that there are two 30S intermediates, p130S and p230S, in vivo. These intermediates sediment around 21S and 30S, correspondingly. Also, three 50S intermediates, p150S, p250S, and p350S sediment at 32S, 43S and 50S, respectively. At least five nucleases are responsible to synthesizethe 16S, 23S, and 5S rRNA from one primary transcript in vivo. RNase III, RNase E, RNase G and RNase T are some of thenucleases thatprocessed the syntheses of the 16S, 23S, and 5S  one primary transcript. The primary transcript is cleaved by RNase III to produce precursor 16S rRNA, precursor 23S rRNA, and precursor 5S rRNA. RNase E further cleaves the 5′ end to produce 66 extra nucleotides at the 5′ end. The 5′ end next cleaved by RNase G. Without RNase E, this procedure could occur, but in the much slower rate. Finally, an unknown RNase causes final maturation at the 3′ end.

Comparing Ribosome Assembly In Vitro and In Vivo

In vivo the reconstitution reaction for ribosome assembly happens much faster than assembly in vitro.The activation energy for assembly is higher in vivo. In vivo, reconstitutions have revealed that ribosomal assembly significantly occurs faster in the presence of the various cofactors. The rRNA components for in vivo and in vitro intermediates are different. In vivoone primary transcript is required to synthesize the 16S, 23S, and 5S rRNA. However, in vitro ribosomal assembly just required fully processed rRNA. The composition of ribosomal protein in 30S and 50S are not identical, but they are similar in both vivo and vitro. A large RNA is able to increaseits lengtheasily. Therefore, the possibility of misfolding is in high rang, and it is so stable. Thus, one of the reasons that causes ribosome assembly be much slower in vitro than in vivo is misfolding of RNA structures. However, there is a solution for this problem, and it is the existence of ribosome assembly factors. They can possibly help limit the misfolding by simplifying proper rRNA folding and protein-RNA interfaces. Also, another function of these factors is to serve as sensors of check points through the process of the ribosomal assembly.

This is about fifty years that researchers study about the assembly of the ribosome, and they work on them about several decades. This research is essential and necessary because assembled ribosome has been involved in several human diseases. Therefore, knowing about the assembly of ribosome may lead to new helpful cures.

Citation

Mangiarotti G, Apirion D, Schlessinger D, Silengo L. 1968. Biosynthetic precursors of 30S and 50S ribosomal particles in Escherichia coli. Biochemistry (61-65).

DNA ligase


On the lagging strand of the replication fork a outcome of broken DNA synthesis is formed by interruptions in the phosphodiester backbone of DNA, but it will be fixed by DNA ligases. Thus, DNA ligase is used for joining the Okazaki fragments after DNA pol I has acted.

nucleotidyltransferases (NTases) which are DNA ligases use either NAD+ or ATP to catalyze phosphodiester bond formation. According to the article Eukaryotic DNA Ligases: Structural and Functional Insights, in bacteria, archaea, and viruses DNA ligases have been classified both ATP- and NAD+-dependent; however, ATP-dependent DNA ligases have been identified in eukaryotes.

Lately, genome sequencing has discovered that archaeal and prokaryotic organisms also have multiple DNA ligases. Now, there will be explained the three families DNA ligases in eukaryote, the roles of their cellular, and how some of human disease are caused by imperfection in DNA ligation.

DNA ligases is an enzyme that catalyzes the reaction in three steps, and it is energetically favorable. In first step, AMP, adenosine 5′-monophosphate, is transferred to lysine in an active-site. After that, it will be moved to the end of DNA where the 5′-PO4 is. Finally, at the last step, a following DNA from the 3′-OH part of strand hits the 5′-PO4 to free the adenosine 5′-monophosphate and makes the production. In eukaryotic and archaeal, an ATP cofactor is used by DNA ligases, whereas in most bacteria NAD+ is used by DNA ligases.

In human, Escherichia coli and Chlorella virus PCBV-1, the complex structure of DNA ligases and DNA are so similar, and it is like a ring. The DNA ligases have three domains in their catalytic parts. The three domains are NTase, OB-fold, and another domain which its name is different in E-coli, human and virus DNA ligases. NTase and OB-fold domain join 3′and 5′ ending parts of DNA. However, the third domain in DNA ligases makes the enzyme look like the ring, and completes the structure of the enzyme. Therefore, the enzyme can easily bind to DNA. According to Eukaryotic DNA Ligases: Structural and Functional Insights the names of the third domain in DNA ligases of virus, bacteria, and human are mentioned in this paragraph. “The Chlorella ligase has an insertion in the OB-fold domain, termed the latch, which is disordered in the absence of DNA and bridges between the two domains of the catalytic core. E. coli LigA has a C-terminal helix-hairpin-helix (HhH) domain engaging the DNA that is functionally analogous to the N-terminal DNA-binding domain (DBD) of human DNA ligase I.”

DNA Ligase I Family

The CDC9 are genes that code the Saccharomyces cerevisiae. This gene is very aware of temperature change. The alleles are super responsive to the temperature change. As soon as temperature changed the DNA will be hurt. Therefore, the CDC9 has DNA ligases to rejoin the DNA. The human have LIG1 gene that is homologous to CDC9 gene. The CDC9 gene codes a polypeptide for DNA ligase, but the LIG1 does not. This article mentioned, “The conditional lethal phenotype of a cdc9 mutant strain was utilized to identify cDNAs, encoding human DNA ligase I, that permitted growth at the nonpermissive temperature. Unlike the yeast CDC9 gene, the human LIG1 gene does not encode a polypeptide that is targeted to mitochondria.”

The reason that CDC9 is a very important gene in S. cerevisiae, is because the deactivation of LIG1 gene in mammals, causes of the cell death. Only if the cell has DNA ligase I in embryonic cell, the cell will not die. In addition, DNA ligase I is very important for embryonic stem cell switches normally to liver. However, it is not vital for replication in cell of mammals.

DNA Ligase III Family

Only vertebrates have the LIG3 gene. Therefore, genetically good lower eukaryotes like S. cerevisiae do not able to define the roles of LIG3 gene within the cells. Furthermore, because of their bigger range of DNA ligases, the yeast homology with LIG1 and LIG4 of mammals cannot be compared to cells of mammal. Three different DNA ligase polyeptides are produced by The LIG3 gene of eukaryotes. As the article claimed, “Nuclear and mitochondrial versions of DNA ligase IIIα are ubiquitously synthesized from DNA ligase IIIα mRNA by an alternative translation initiation mechanism. In addition, a germ cell-specific alternative splicing mechanism, in which the terminal 3′-coding exon in DNA ligase IIIα mRNA is replaced by a different exon, generates DNA ligase IIIβ mRNA.” Therefore, DNA ligase IIIα mRNA synthesizes nuclear and mitochondrial translation of DNA ligase IIIα via a different translation mechanism.

DNA Ligase IV Family

Finally, this article explains that “The polypeptides encoded by the human LIG4 and the yeast DNL4 genes have an N-terminal catalytic domain with a C-terminal extension that contains tandemly arrayed BRCT motifs”. Thus, the human LIG4 and the yeast DNL4 genes both encoded the polypeptides that have an N-terminal that is catalytic core and a C-terminal addition that has cycle to BRCT patterns. Before of DNA ligase IV cloning, the detection of DNA ligase activity is failed. The reason for this failure is that characteristics come together with the similar size of the polypeptides encoded by the LIG3 and LIG4 genes. Also, DNA ligase IV can combine DNA ends that are not complementary. The reason that the enzyme has this ability is because of disparity and small gaps. This feature of DNA ligase IV can tell apart it from DNA ligases I and III. Also, in the lack of protein XRCC4, DNA ligase IV is not stable.

In conclusion, the study of DNA ligases is interesting because it is necessary for joining of DNA strands in replication and fixing. Recently scientists have important approaching into the molecular mechanism of the three steps of the ligation reaction. This information about the arrangement and understanding about the cellular roles of human DNA ligases, caused the progress in the research area about DNA ligase. Hopefully, these developments will one day become valuable for cancer therapeutics.

Citation

Ellenberger Tom, E. Tomkinson Alan. Eukaryotic DNA Ligases: Structural and                                                                         Functional Insights, Department of Biochemistry and Molecular Biophysics,  Washington University School of Medicine, St. Louis, Missouri.