Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Homologous recombination wikipedia , lookup
Eukaryotic DNA replication wikipedia , lookup
United Kingdom National DNA Database wikipedia , lookup
DNA nanotechnology wikipedia , lookup
Microsatellite wikipedia , lookup
DNA replication wikipedia , lookup
DNA polymerase wikipedia , lookup
THE CENTRAL DOGMA OF MOLECULAR BIOLOGY Jony Mallik B. Pharmacy; M. Pharmacy E-mail: [email protected] INTRODUCTORY PRINCIPLE Biomolecules are the molecules that are synthesized within living organism and perform different functions in that organism in terms of separate metabolic & biosynthetic purposes. The biomolecules could be a coarse one like- polypeptides, polysaccharides, lipids or any other macromolecules. Protein is the only biomolecule which is synthesized depending on body individual need & participate in different biologic signaling system like plasma protein, membrane protein, receptor protein, enzyme protein system. Proteins are the essential tools for proper growth & repair of muscle. Some proteins, people may easily get from food stuff but, some are very authentic & body usually biosynthesized that types of protein. In vivo protein synthesis and its phenomenon is the central dogma of molecular biology. Central dogma of molecular biology is the explanation of two different steps required in synthesizing a protein. Central dogma deals with the details of genetic information (DNA) transformation to m RNA & then to a protein. The two vulnerable steps of protein synthesis are Transcription Translation Deoxyribonucleic acid (DNA) DNA is the molecule of life & a very essential nucleic acid located at the chromosome of cellular nucleus. All the genetic information is stored in DNA molecule & transformed into mRNA (Transcription) then to a protein (Translation), that is why DNA is sometimes said as “The Reserve Bank of Genetic Information”. In DNA there are four bases: adenine (A), guanine (G), thymine (T) and cytosine (C). Adenine and guanine are purines; thymine and cytosine are pyrimidines. A nucleoside is a pyrimidine or purine base covalently bonded to a sugar. In DNA, the sugar is deoxyribose and so this is a deoxynucleoside. There are four types of deoxynucleoside in DNA; deoxyadenosine, deoxyguanosine, deoxythymidine and deoxycytidine. A nucleotide is base + sugar + phosphate covalently bonded together. In DNA, where the sugar is deoxyribose, this unit is a deoxynucleotide. In DNA the nucleotides are covalently joined together by 3’5’phosphodiester bonds to form a repetitive sugar–phosphate chain which is the backbone to which the bases are attached. The DNA sequence is the sequence of A, C, G and T along the DNA molecule which carries the genetic information. In a DNA double helix, the two strands of DNA are wound round each other with the bases on the inside and the sugar–phosphate backbones on the outside. The two DNA chains are held together by hydrogen bonds between pairs of bases; adenine (A) always pairs with thymine (T) and guanine (G) always pairs with cytosine (C). DNA Replication DNA replication is the process of the genesis of two identical replica of the parent DNA molecule under the agency of some sorts of enzyme. Newly formed molecule contains same genetic information that the parent molecule may have. Replication process involve numerous complicated task. All the new molecule further participate in transcription process. Enzymes & Protein that play a key role in DNA replication process are DNA polymerase I & III DNA primase DNA gyrase & nuclease DNA helicase DNA ligase Single stranded DNA binding protein (SSB) DNA Replication models The process of DNA Replication was hiding many secrets. One of the most important was how the two daughter strands are created. As we have noticed in previous chapters of our site the DNA is a complex of two chains! In order the hereditary phenomenon to be explained, these strands should be accurately copied and transmitted from the parental cell to the daughter ones. These are three possible models that describe the accurate creation of the daughter chains: Semiconservative Replication According to this model, DNA Replication would create two molecules. Each of them would be a complex of an old (parental and a daughter strand). Conservative Replication According to this model, the DNA Replication process would create a brand new DNA double helix made of two daughter strands while the parental chains would stay together. Dispersive Replication According to this model the Replication Process would create two DNA double-chains, each of them with parts of both parent and daughter molecules. Replication Process The overall DNA replication process is very complicated job and involves a set of proteins and enzymes that collectively assemble nucleotides in the predetermined sequence. A particular replication process is consists of following distinct steps to form two identical replica. INITIATION ► The site from which the replication starts are called Replication origin or Origin of replication. In order for DNA replication to begin, the double stranded DNA helix must open, for that both of the helicase & SSB protein bind to that region to unwind the helix & stabilize the DNA into two strand. ► The open portion of parent DNA are referred as “ Replication fork”, which is asymmetrical as the two single strands run in a anti-parallel direction. PRIMER SYNTHESIS ► The synthesis of a new, complementary strand of DNA using the existing strand as a template is brought about by enzymes known as DNA polymerases. In addition to replication they also play an important role in DNA repair and recombination. ► One of the most important steps of DNA Replication is the binding of RNA Primase in the initiation point of the 3'-5' parent chain. RNA Primase can attract RNA nucleotides which bind to the DNA nucleotides of the 3'-5' strand due to the hydrogen bonds between the bases. RNA nucleotides are the primers (starters) for the binding of DNA nucleotides. SYNTHESIS OF LEADING STRAND ► Then DNA polymerase III play its role in initiating the leading strand which required few steps & therefore is synthesized quickest. ► DNA polymerase III (DNA pol III) recognizes the 3' OH end of the RNA primer, and adds new complementary nucleotides. As the replication fork progresses, new nucleotides are added in a continuous manner, thus generating the new strand. SYNTHESIS OF LAGGING STRAND ► DNA is synthesized in a discontinuous manner by generating a series small fragments of new DNA in the 5' → 3' direction. These fragments are called Okazaki fragments, which are later joined to form a continuous chain of nucleotides. This strand is known as the lagging strand since the process of DNA synthesis on this strand proceeds at a lower rate. LIGATION & TERMINATION ►After primer removal is completed the lagging strand still contains gaps or nicks between the adjacent Okazaki fragments. The enzyme ligase identifies and seals these nicks by creating a phosphodiester bond between the 5' phosphate and 3' hydroxyl groups of adjacent fragments. ► This replication machinery halts at specific termination sites which comprise a unique nucleotide sequence. This sequence is identified by specialized proteins called tus which bind onto these sites, thus physically blocking the path of helicase. When helicase encounters the thus protein it falls off along with the nearby single-strand binding proteins. DIAGRAMMATIC SEQUENCES OF DNA REPLICATION Initiation Primer synthesis Synthesis of leading strand Synthesis of lagging strand Termination TRANSCRIPTION Transcription is the preliminary step of the gene expression that deals with the details of the synthesis on mRNA (messenger RNA) from the promoter gene on the DNA molecule under the agency of some enzymes & transcription factors. The enzymes involved in transcription are called RNA polymerases. Prokaryotes have one type; eukaryotes have three types of nuclear RNA polymerases. RNA Synthesis There are a number of different types of RNA, which play different roles in the cell: ♦ mRNA - encodes proteins. ♦ rRNA - forms the ribosome, including the active site for peptide bond formation. ♦ tRNA - adaptor, binds amino acids and rRNA and translates between mRNA and protein. ♦ snRNA - small nuclear RNA, forms snRNPs, which process mRNA by removing introns. ♦ snoRNA - small nucleolar RNA, forms snoRNPs, which process rRNA, mostly by methylation and isomerisation. ♦ si RNA - small interfering RNA, involved in gene silencing and regulation. ♦ gRNA - guide RNA, needed for RNA editing, the removal and insertion of bases into mRNA ♦ hnRNA - rag-bag of unprocessed pre-mRNA transcripts and other heterogeneous nuclear RN As of less well defined function. RNA polymerase ► RNA polymerase is the enzyme that generates RNA from DNA. Cells contain 20 times more RNA than DNA: in fact, about 5% of the cell is RNA, although only 5% of this 5% is mRNA, because most of the RNA in the cell is rRNA. ► Since the majority of RNA is rRNA, Significantly more RNA is transcribed than translated. This is especially true in eukaryotes, whose mRNA requires processing to remove introns. ► The primary gene products of RNA polymerase (in eukaryotes) are: • (pre-) mRNA (messenger); • rRNA (ribosomal); • tRNA (transfer); • snRNA (small nuclear - spliceosomes); • Other hnRNA (heterogeneous nuclear, such as snoRNA – small nucleolar). Eukaryotic RNA Polymerases Eukaryotes have three RNA polymerases which synthesized different type of RNA RNA polymerase I - rRNA. RNA polymerase II - mRNA. RNA polymerase III - tRNA Steps Involve in Transcription of DNA to mRNA The overall transcription process required three distinct steps to complete Initiation Elongation Termination Initiation Transcription actually starts from a very special region of DNA double helix called “Promoter Region”, a region which has meaningful nucleobase sequences of gene product (Protein). RNA polymerase with transcription factor (sigma factor) associates to the promoter region to initiate transcription Promoters 1. Only one strand of the DNA that encodes a promoter, a regulatory sequence, or a gene needs to be written. 2. The strand that is written is the one that is identical to the RNA transcript, thus the antisense strand of the DNA is always selected for presentation. 3. The first base on the DNA where transcription actually starts is labeled +1. 4. Sequences that precede, are upstream of the first base of the transcript, are labeled with negative numbers. Sequences that follow the first base of the transcript, are downstream, are labeled with positive numbers. Steps of Transcription Process Elongation RNA polymerase starst to move forward in a 5' to 3' direction. The polymerase induces the 3' hydroxyl group of the nucleotide at the 3' end of the growing RNA chain which attacks (nucleophilic) a phosphorous of the incoming ribonucleotide. The complementary sequence of DNA come out as pre-mRNA during movement of RNA polymerase Termination The mechanisms by which eukaryotes terminate transcription are poorly understood. Most eukaryotic genes are transcribed for upto several thousand base pairs beyond the actual end of the gene. The excess RNA is then cleaved from the transcript when the RNA is processed into its mature form. Finally the sigma factor & RNA polymerase remains separate as intact form. Processing of RNA/ RNA splicing The newly synthesized pre-mRNA contains “exon” & “intron” segment within it, where the intron part has no impact on protein synthesis hence, a further processing need to remove intron & make it as pure & mature m RNA. The process of cutting & removing introns from pre-mRNA & joining the exons is referred as “RNA splicing” RNA processing Mechanism of RNA processing / splicing The mechanism of RNA splicing is very complicated. The biochemical mechanism of splicing consists of two reactionsFirst Reaction In order to start the first reaction, the ending nucleotide of the intron react to first nucleotide to form intron lariat, which will be remove at second reaction. Second Reaction Formed intron lariat then cut & removed from the pre-mRNA & exons are joined together to form the mature or spliced RNA. Mechanism of RNA splicing TRANSLATION Translation is a very key portion of central dogma, that deals with the synthesis of gene product (protein) form spliced mRNA by using tRNA, ribosomal subunit & some factors. Steps Involve in Translation of mRNA to Protein The overall mechanism of protein synthesis in eukaryotes is basically the same as in prokaryotes, with three phases defined as initiation, elongation and termination. However, there are some significant differences, particularly during initiation. Translation of mRNA into a protein requires ribosomes, mRNA, tRNA, exogenous protein factors and energy in the form of ATP and GTP. Initiation Four major steps are required to initiate translation: ribosome dissociation, formation of a pre-initiation complex, formation of the 405 initiation complex and formation of the 805 initiation complex. The first step is the formation of a pre-initiation complex consisting of the 40S small ribosomal subunit, Met-tRNAi met, eIF-2 and GTP; The pre-initiation complex now binds to the 5’ end of the eukaryotic mRNA, a step that requires eIF-4F (also called cap binding complex) and eIF-3. The complex now moves along the mRNA in a 5’ to 3’ direction until it locates the AUG initiation codon. Once the complex is positioned over the initiation codon, the 60S large ribosomal subunit binds to form an 80S initiation complex, a step that requires the hydrolysis of GTP and leads to the release of several initiation factors. Elongation The elongation stage of translation in eukaryotes requires three elongation factors, eEF-1A, eEF-IB and eEF-2, which have similar functions to their prokaryotic counterparts EF-Tu, EF-Ts and EF-G. Although most codons encode the same amino acids in both prokaryotes and eukaryotes, the mRNAs synthesized within the organelles of some eukaryotes use a variant of the genetic code. During elongation the protein is synthesized one amino acid at a time on the 80S ribosome. This process occurs in three major steps: binding of charged tRNA, peptide bond formation, translocation of the growing peptide chain. Termination When a stop codon appears at the A site translation is terminated. There are no tRNA's that recognize stop codons. Instead releasing factors, eRF, recognize the stop codon. The releasing factors along with peptidyl transferases and GTP catalyze the hydrolysis of the bond between the polypeptide chain and the tRNA. The protein and tRNA disassociate from the P site and the ribosome dissociates into the 405 and 605 subunits releasing the mRNA. Diagrammatic sequence of Protein synthesis References 1) David Hames., Niger Hooper., Biochemistry- 3rd edition. 2) Jeremy M. Berg.,John L. Tymoczko., Lubert Stryer. Biochemistry-5th edition. 3) Robert K. Murray., Daryl K. Granner., Peter A. Mayes., Victor W. Rodwell., Harper’s Illustrated Biochemistry- 22nd edition. 4) H.P.Gajera., S.V.Patel., Fundamentals of Biochemistry-A Textbook- 1st edition (2008). 5) www.google.com (Wikipedia) THE END