Gene expression The flow of genetic information DNA RNA Protein The genetic information flows from DNA to mRNA and then to the protein synthesizing machinery The synthesis of RNA molecule from DNA is called transcription All eukaryotic cells have five major classes of RNA 1 Ribosomal RNA rRNA 2 Messenger RNA mRNA 3 Transfer RNA tRNA 4 Small nuclear RNA snRNA 5 microRNA miRNA NOTE first 3 are used in protein synthesis The small RNAs are involved in mRNA splicing 1 General steps of initiation elongation and termination with 5 to 3 polarity and regulation of gene expression Similarities between replication and transcription 2 Large multicomponent initiation complexes 3 Adherence of Watson Crick base pairing rules Differences between replication and transcription 1 Ribonucleotides are used in RNA synthesis rather than deoxy ribonucleotides 2 U replaces T as the complementary base for A in RNA 3 A primer is not involved in RNA synthesis 4 Only a portion of genome is transcribed or copied to RNA whereas must be copied during DNA replication 5 There is no proofreading function during RNA transcription Strand that is transcribed or copied into RNA molecule is termed template strand of the Template strand DNA Other DNA strand non template is referred to as the coding strand of that gene Information in the template strand is read out in the 3 to 5 direction 1 Sequence of ribonucleotides in template is complementary to the sequence of deoxy ribonucleotides in the template strand of the double stranded DNA molecule In the coding strand complementary strand the sequence is same as that of the sequence of nucleotides in the primary transcript Transcription Unit Defined as region of DNA that includes the signals for transcription initiation elongation and termination DNA dependent RNA polymerase is the enzyme responsible for the polymerization of ribonucleotides into a sequence complementary to the template strand of the gene The enzyme attaches at a specific site the promoter on the template strand This is followed by initiation of RNA synthesis at the starting point and the process continues until a termination sequence is reached Transcriptional unit and primary transcript Primary transcript transcript identical The RNA product which is synthesized in the 5 to 3 direction is the primary In prokaryotes this can represent the product of several contiguous genes In mammalian cells it usually represents the product of a single gene The 5 terminals of the primary RNA transcript and the mature cytoplasmic RNA are The starting point of transcription corresponds to the 5 nucleotide of the mRNA This is designated position 1 as is the corresponding nucleotide in the DNA The numbers increase as the sequence proceeds downstream 2 The nucleotide in the promoter adjacent to the transcription initiation site is designated These negative numbers increase as the sequence proceeds upstream away from the This provides a conventional way of defining the location of regulatory elements in the 1 initiation site promoter Transcription unit The process of transcription of a typical gene of E Coli can be divided in to three phases Prokaryotic transcription Steps of RNA Synthesis i Initiation ii Elongation iii Termination i Initiation of Transcription highly conserved Pribnow box left of the transcription start site 35 Sequence Initiation of transcription involves the binding of the RNA polymerase holoenzyme to the promoter region on the DNA to form a preinitiation complex or PIC Characteristic Consensus nucleotide sequence of the prokaryotic promoter region are This is a stretch of 6 nucleotides 5 TATAAT 3 centered about 8 10 nucleotides to the A second consensus nucleotide sequence 5 TTGACA 3 is centered about 35 bases to the left of the transcription start site 3 Binding of RNA polymerase RNAP to the promoter region is followed by a conformational change of the RNAP and the first nucleotide almost always a purine then associates with the initiation site on the subunit of the enzyme In the presence of the appropriate nucleotide RNAP catalyzes the formation of a phosphodiester bond and the nascent chain is now attached to the polymerization site on In both prokaryotes and eukaryotes a purine ribonucleotide is usually the first to be the subunit of RNAP polymerized into the RNA molecule After 10 20 nucleotides have been polymerized RNAP undergoes a second conformational change leading to promoter clearance Once this transition occurs RNAP physically moves away from the promoter transcribing down the transcription unit leading to the next phase of the process elongation II Elongation step of Transcription As the elongation complex containing the core RNA polymerase progresses along the DNA molecule DNA unwinding must occur in order to provide access for the appropriate base pairing to the nucleotides of the template strand The extent of this transcription bubble i e DNA unwinding is constant throughout and is about 20 base pairs per polymerase molecule RNA polymerase has associated with it an unwindase activity that opens the DNA helix Topo isomerase both precedes and follows the progressing RNAP to prevent the formation of super helical complexes Base pairing rule is followed during the incorporation of ribonucleotides III Termination of transcription 4 Termination of the synthesis of the RNA molecule in bacteria is of two types a Rho dependent termination The termination process is signaled by a sequence in the template strand of the DNA molecule a signal that is recognized by a termination protein the rho factor Rho is an ATP dependent RNA stimulated helicase that disrupts the nascent RNA DNA complex b Rho independent termination This process requires the presence of intra chain self complementary sequences in the newly formed primary transcript so that it can acquire a stable hair pin turn that slows down the progress of the RNA polymerase and causes it to pause temporarily Near the stem of the hairpin a sequence occurs that is rich in G and C This stabilizes the secondary structure of the hair pin Beyond the hair pin the RNA transcript contains a strings of Us the bonding of Us to the This facilitates the dissociation of the primary transcript from DNA After termination of synthesis of the RNA molecule the enzyme separates from the DNA corresponding As is weak template With the assistance of another factor the core enzyme then recognizes a promoter at which the synthesis of a new RNA molecule commences