Your browser is too old
We can't provide a great video experience on old browserUpdate now
Three Roles of RNA in Translation
This Sqadia video is the demonstration of Basic Molecular Genetic Mechanisms - II. In eukaryotic cells, protein synthesis occurs in the cytoplasm, where three types of RNA molecules come together to perform different but cooperative functions. The genetic code used by cells is a triple code, with every three-nucleotide sequence, or codon, being “read” from a specified starting point in the mRNA. The sequence of codons that runs from a specific start codon to a stop codon is called a reading frame. This precise linear array of ribonucleotides in groups of three in mRNA specifies the precise linear sequence of amino acids in a polypeptide chain and signals where synthesis of the chain starts and stops. In most mRNAs, the start codon specifying this amino terminal methionine is AUG. The function of tRNA molecules depends on their precise 3D structures. Some 30–40 different tRNAs have been identified in bacterial cells and as many as 50–100 in animal and plant cells. Thus, the number of tRNAs in most cells is more than the number of amino acids used in protein synthesis (20) and differs from the number of amino acid codons in the genetic code (61). The capability of a single tRNA anticodon to recognize more than one, but not necessarily every, codon corresponding to a given amino acid. This broader recognition can occur because of nonstandard pairing between bases in the so-called wobble position. In decoding the genetic message, the first step is attachment of the appropriate amino acid to a tRNA, catalyzed by a specific aminoacyl-tRNA synthetase. A ribosome is composed of three (in bacteria) or four (in eukaryotes) different rRNA molecules and as many as 83 proteins, organized into a large subunit and a small subunit. During translation, a ribosome moves along an mRNA chain, interacting with various protein factors and tRNAs and very likely undergoing large conformational changes.
Ribosome assembles, complexed with a mRNA and an activated initiator tRNA, which is correctly positioned at the start codon. During translation initiation, the 5 cap of an mRNA to be translated is bound by the eIF4E subunit of the eIF4 cap binding complex. The initiation complex then probably slides along, or scans, the associated mRNA as the helicase activity of eIF4A uses energy from ATP hydrolysis to unwind the RNA secondary structure. Scanning stops when the tRNAi Met anticodon recognizes the start codon, which is the first AUG downstream from the 5 end in most eukaryotic mRNAs. Recognition of the start codon leads to hydrolysis of the GTP associated with eIF2, an irreversible step that prevents further scanning. A set of special proteins, termed elongation factors (EFs), are required to carry out the process of chain elongation. The key steps in elongation are entry of each succeeding aminoacyl-tRNA, formation of a peptide bond, and the movement, or translocation, of the ribosome one codon at a time along the mRNA. Then, a newly synthesized protein folds into its native 3D conformation, facilitated by proteins called chaperones. Additional release factors then promote dissociation of the ribosome, freeing the subunits, mRNA, and terminal tRNA for another round of translation.
Definitive evidence that duplex DNA is replicated by a semiconservative mechanism came from a now classic experiment conducted by M. Meselson and W. F. Stahl. The unwinding of the parental DNA strands is by specific helicases, beginning at unique segments in a DNA molecule called replication origins, or simply origins. Local unwinding of duplex DNA produces torsional stress, which is relieved by topoisomerase I. RNA polymerase can find an appropriate transcription start site on duplex DNA and initiate the synthesis of an RNA complementary to the template DNA strand. With a primer base-paired to the template strand, a DNA polymerase adds deoxynucleotides to the free hydroxyl group at the 3 end of the primer as directed by the sequence of the template strand. The formation of Leading strand continuously takes place in 3' to 5'direction, whereas the formation of lagging strand in not continuous. Each of the primers, base-paired to the template strand, is elongated in the 5->3 direction, forming discontinuous segments called Okazaki fragments. Finally, an enzyme called DNA ligase joins the adjacent fragments. The general consensus is that all prokaryotic and eukaryotic cells employ a bidirectional mechanism of DNA replication. In the case of SV40 DNA, replication is initiated by binding of two large T-antigen hexameric helicases to the single SV40 origin and assembly of other proteins to form two replication forks. Unlike SV40 DNA, eukaryotic chromosomal DNA molecules contain multiple replication origins separated by tens to hundreds of kilobases.
Viruses: Parasites of the Cellular Genetic System
Viruses cannot reproduce by themselves and must commander a host cell’s machinery to synthesize viral proteins and in some cases to replicate the viral genome. In addition to their obvious importance as causes of disease, viruses are extremely useful as research tools in the study of basic biological processes. The surface of a virion contains many copies of one type of protein that binds specifically to multiple copies of a receptor protein on a host cell. A virus that infects only bacteria is called a bacteriophage, or simply a phage. The nucleic acid of a virion is enclosed within a protein coat, or capsid, composed of multiple copies of one protein or a few different proteins, each of which is encoded by a single viral gene. A capsid plus the enclosed nucleic acid is called a nucleocapsid. The number and arrangement of coat proteins in icosahedral, or quasi-spherical, viruses differ somewhat depending on their size. In many DNA bacteriophages, the viral DNA is located within an icosahedral “head” that is attached to a rod like “tail.” The phospholipids in the viral envelope are similar to those in the plasma membrane of an infected host cell. The number of infectious viral particles in a sample can be quantified by a plaque assay.
Life Cycle of Viruses
In lytic cycle, DNA genome is transported into the cell nucleus. Once inside the nucleus, the viral DNA is transcribed into RNA by the host’s transcription machinery. Processing of the viral RNA primary transcript by host cell enzymes yields viral mRNA, which is transported to the cytoplasm and translated into viral proteins by host-cell ribosomes, tRNA, and translation factors. Assembly of the capsid proteins with the newly replicated viral DNA occurs in the nucleus, yielding hundreds to thousands of progeny virions. In lysogenic cycle, some retroviruses contain cancer-causing genes (oncogenes), and cells infected by such retroviruses are oncogenically transformed into tumor cells. Among the known human retroviruses are human T-cell lymphotrophic virus (HTLV), which causes a form of leukemia, and human immunodeficiency virus (HIV), which causes acquired immune deficiency syndrome (AIDS). Both of these viruses can infect only specific cell types, primarily certain cells of the immune system and, in the case of HIV, some central nervous system neurons and glial cells. Only these cells have cell-surface receptors that interact with viral envelope proteins, accounting for the host-cell specificity of these viruses. Unlike most other retroviruses, HIV eventually kills its host cells. The eventual death of large numbers of immune-system cells results in the defective immune response characteristic of AIDS.