Transcription PDF

Transcription Dr Safa Hamid OBJECTVE By the end of lecture the student will be able to describe: 1.Describe RNA Structure and nucleotides entering in its structure. 2.Recognize RNA Synthesis and steps of transcription. 3.Explain posttranscriptional modification Transcription Transcr...

Transcription Dr Safa Hamid OBJECTVE By the end of lecture the student will be able to describe: 1.Describe RNA Structure and nucleotides entering in its structure. 2.Recognize RNA Synthesis and steps of transcription. 3.Explain posttranscriptional modification Transcription Transcription : is the process of synthesis of RNA from DNA template by DNA- dependent RNA polymerase. Before discussion of gene transcription one must know the following: 1. Gene : It is a linear sequence of nucleic acids that encode proteins and structural RNA. Gene sequences are written from 5’ to 3’ end. 2Gene expression : is the process by which information from a gene is used in the synthesis of gene products which are usually proteins by protein coding genes and RNA by non-protein coding genes.. 3Eukaryotic genes are composed of coding exons, non coding introns and non coding recognition markers called consensus sequence.. 4 Gene expression starts by transcription. Types of RNAs in Prokaryotes There are three types of RNAs, found in both prokaryotic and eukaryotic cells; these are: 1. mRNA 2. rRNA 3. tRNA  Features of transcription 1. Only one strand of the DNA strands is transcribed. This strand is called template strand (antisence strand or non coding strand), because it provides template for ordering the sequence of nucleotides in an RNA transcript. 2. The other strand is called coding, nontemplate or sense strand, because its sequence is the same as the newly synthesized RNA transcript. 3. The template is copied according to base pairing role, in which G binds to C and A binds to U instead of T( in DNA)  -4The RNA, then, is complementary to the DNA template (antisense) strand and identical to the coding (sense) strand, with U replacing T..The template is copied as it is in DNA synthesis, in which a G on the DNA specifies a C in the RNA, a C specifies a G, a T specifies an A, but an A specifies a U instead of a T.. 5 RNA is synthesized from its 5'-end to its 3'- end, antiparallel to its DNA template strand Template dependant process Coding strand Template strand TRANSCRIPTION STAGES INITIATION ELONGATION TERMINATION Transcription Requirements: Specific DNA sequences to be copied (gene) RNA polymerases. Several protein factors.  The Role of DNA in Transcription The DNA strand that transcribed into an RNA is the template strand. The complementary strand is the coding strands.  The gene is the DNA segment that encodes a functional polypeptide. The transcriptional unit; is the DNA region that include the signals for initiation, elongation and termination of a gene.  The RNA polymerase Enzyme Transcription is catalyzed by multimeric enzymes known as RNA-polymerases. RNA polymerases are large multimeric proteins, consisting of several subunits.  Classes of RNA Polymerases In prokaryotic cells, all types of RNA are synthesized by a single RNA-polymerase. Eukaryotic cells have three distinct, RNA polymerases (pol) I, II and III. Each polymerase synthesize a different class of RNA.  Prokaryotic RNA polymerase Prokaryotes RNA polymerase is a multisubunit enzyme has the following functions: A-Recognizes a nucleotide sequence (the promoter region) at the beginning of the transcription unit. b-Makes a complementary RNA copy of the DNA template strand. C- Recognizes the end of the DNA sequence to be transcribed (the termination region). Polymerase synthesizes all types of RNA except for the short RNA primers needed for DNA replication Prokaryotic RNA polymerase RNA polymerase is a holoenzyme formed of 1. Core enzyme: consists of 4 subunits (α2 β β-) two identical (2α) subunits, two non identical β subunits ( 1β, and 1β'),.They are required for: a. Structural component of the enzyme (2α). b. Template binding (β'). C -5' → 3' RNA polymerase activity (β). 2-Protein part called Sigma σ factor. The σ subunit (―sigma factorǁ) enables RNA polymerase to recognize promoter regions on the DNA. Both core enzyme and the sigma factor form the holoenzyme. Promoter area : It is DNA sequences that select or determine that the start site of RNA synthesis.It is the binding site for RNA polymerase holoenzyme. It is not transcribed. The promoter region is recognized by RNA polymerase sigma factor. A base in the promoter region is assigned a negative number if it occurs prior to the transcription start site (to the left of, toward the 5'-end of, or ―upstreamǁ of sence strand).The first base at the transcription start site is assigned a position of +1 There is no base designated (0) Promoter region Promoter region in prokaryotes is consists of : 1. TATA box (Pribnow box): It is formed of six nucleotides (TATAAT) and is located -10 bases upstream to the start transcription point (+1 point). It is the site of initial DNA melting (unwinding). Melting of a short stretch (about 14 bases) leads to formation of transcription bubble. 2. The (TTGACA) box: It is located -35 bases upstream to the start point. This box is the initial point of contact for the holoenzyme.  The RNA product which is synthesized in the 5’ to 3’ direction is called primary transcript. The DNA template strand is read in 3’ to 5’ by RNA polymerase enzyme and the new mRNA is synthesized in the direction of 5’ to 3’ The bacterial promoter contains specific regulatory elements: Transcription in prokaryotes Steps of transcription : It occurs in the cytoplasm. The process of transcription in prokaryotes can be divided into three phases: 1. Initiation. 2. Elongation. 3. Termination  1- Initiation Initiation of transcription includes the following steps : A- The sigma subunits enables the RNA polymerase to recognize the promoter sites. B-Holoenzyme form a closed complex with the (5'TTGACA-3') sequence. C- The holoenzyme moves to the (5'-TATAAT-3'), centered at about –10 (Pribnow box). Local unwinding and opening of the DNA helix occurs  Elongation it include the following : 1. RNA polymerase begins to synthesize a transcript of the DNA sequence.The first nucleotide to be transcribed (start site) is denoted as (+1) it is usually A (adenine), the second is denoted (+2). 2. The elongation phase is said to begin when the transcript (typically starting with a purine) exceeds ten nucleotides in length. 3. Sigma is then released, and the core enzyme is able to leave (―clearǁ) the promoter and move along the template strand in a processive manner.. Like DNA polymerase.  5. RNA polymerase uses nucleoside triphosphates as substrates and releases pyrophosphate each time a nucleoside monophosphate is added to the growing chain. 6. Transcription is always in the 5'→3' direction.(. In contrast to DNA polymerase, RNA polymerase does not require a primer and does not appear to have proofreading activity). 7. Elongation of the single-stranded RNA chain continues until a termination signal is reached. Transcription Elongation mRNA from DNA, transcription DNA (RNA) A – T(U) G – C(C)  termination It may be 1-1ntrinsic (spontaneous): It is called pindependent termination. 2-p-dependent termination: It dependes upon the participation of a protein known as the ρ (rho) factor. 1 - p - independent termination ( intrinsic or spontaneous ) : The process don’t require ATP. Formation of hairpin loop: newly synthesized (nascent) RNA fold back on itself, forming a GC-rich stem (stabilized by H-bonds) plus a loop due to presence of specific sequence on DNA called palindromic sequence (G-C rich region followed by A-T rich region.  Just beyond the hairpin, the RNA transcript contains a string of Us at the 3'-end. The bonding of these Us to the complementary As of the DNA template is weak. This facilitates the separation of the newly synthesized RNA from its DNA template  2 ) Rho or ρ - Dependent termination: Rho (ρ), is a hexameric adenosine triphosphatase (ATPase) with helicase activity. ρ binds a C-rich ―rho recognition siteǁ near the 3'- end of the nascent RNA and by using its ATPase activity it moves along the RNA until it reaches the termination site then RNA polymerase released from the termination site. The ATP dependent helicase activity of Rho factor, causing the release of the RNA Action of antibiotics: Action of antibiotics: Some antibiotics prevent bacterial cell growth by inhibiting RNA synthesis. For example Rifampin or Rifampicin: It inhibits the initiation of transcription by binding to the β subunit of prokaryotic RNA polymerase, thus interfering with the formation of the first phosphodiester bond.Rifampin is useful in the treatment of tuberculosis  Summary of steps of transcription in procaryotes I - Initiation : 1. RNA polymerase holoenzyme, bind to promoter DNA by sigma factor protein. 2. RNA polymerase creates a transcription bubble, which separates the two strands of the DNA helix. This is done by breaking the hydrogen bonds between complementary DNA nucleotides. II - Elongation : 1.RNA polymerase adds RNA nucleotides to the complementary nucleotides of one DNA strand. 2-.Elongation of the single-stranded RNA chain continues until a termination signal is reached. III - Termination : Termination is done spontaneously or by rho factor protein  Transcription of eukaryotic Transcription of eukaryotic genes is more complicated process than in prokaryotes because : 1-It needs separate polymerases for the synthesis of rRNA, tRNA, and mRNA. 2-In addition, a large number of proteins called transcription factors (TFs) are involved. TFs bind to distinct sites on the DNA—either within the promoter region, close (proximal) to it, or some distance away (distal) from it. Because TFs are encoded by different genes, synthesized in the cytosol, and must transit to their sites of action, they are called trans- acting factors. Transcription factors include TF II A, B, D, E, F & H. In contrast to the holoenzyme of prokaryotes, eukaryotic RNA polymerase II does not itself recognize and bind the promoter  Inhibitors of RNA polymerase II: This enzyme is inhibited by amanitin—a potent toxin produced by the poisonous mushroom. -Amanitin forms a tight complex with the polymerase II, thereby inhibiting mRNA synthesis and, ultimately, protein synthesis.  The promoter region in eukaryotes The promoter region in eukaryotes is formed of the following sequences : 1-The TATA box or Hogness box : (TATAAT) it is located 25 upstream of the transcription start point. It determines where transcription starts. It is recognized by transcription factor TFII D. 2 - The CAAT box (CCAATC) it is located 70 -80 nucleotides upstream of the transcription start site. 3 - the G C box (GGGCGG) : In some genes, no TATA box is typically present. Instead, a GC-rich region (GC box) it is located -40 nucleotide upstream of the transcription start point. These boxes (CAAT box& the G C box) determine the frequency of transcription  Functions of transcription factors are:. 1They help binding of RNA polymerase II to the promoter, formation of a transcription complex at the promoter and initiation of transcription.. 2 Help in the determination of which genes are to be transcribed (Each eukaryotic RNA polymerase has its own promoters and TFs Eukaryotic Transcription Factors STEPS OF TRANSCRIPTION Initiation : RNA Polymerase in prokaryotes is capable of binding directly to promoters, in Eukaryotes this is not possible and is mediated by the transcription factors. 1. TFII D recognizes and binds the TATA box. 2. TFII F brings the polymerase to the promoter. 3. The helicase activity of TFII H melts the DNA and its kinase activity phosphorylates polymerase. 4. Then transcription starts at the starting point  II - Elongation : 1. Polymerase moves downstream, unwinding the DNA and elongating the RNA transcript in th 5’ to 3’ direction with base pairing role.It utilizes nucleotide triphosphate (ATP,GTP, CTP, and UTP) with the release of pyrophosphate. 2. In RNA transcript the sequence is as the coding strand except U instead of T. 3. DNA rewind again.  III - Termination : 1. A specific AAUAAA sequence on mRNA is recognized by termination factors and endonucleases enzyme. These termination factors leads to disossociation of RNA polymerase enzyme and release of the transcript. 2. Dactinomycin (actinomycin D), anticancer drug, used in tumor chemotherapy. binds to the DNA template and interferes with the movement of RNA polymerase along the DNA POST TRANSCRIPTIONAL MODIFICATIONS Eukaryotic RNAs undergo significant post- transcriptional processing. 1-Splicing All 3 classes of RNA are transcribed from genes that contain introns which must be removed The process of intron removal is called RNA splicing. Capping The 5' end of all eukaryotic mRNAs are capped with a unique 5' -----> 5' linkage to a 7-methylguanosine residue. The cap protects mRNA from exonucleases. It is necessary for binding to ribosome for translation. The 5'-Cap of Eukaryotic mRNAs Poly (A) tailing mRNA are also polyadenylated at the 3' end. A stretch of 20 - 250 A residues is then added to the 3' end by the polyadenylate polymerase activity Difference between transcription in prokaryotes and eukaryote PROKARYOTES EUKARYOTE Simple and occurs in Complicated and occurs in cytoplasm the nucleus. Only one Eukaryotic transcription polymerase(holoenzyme) involves separate synthesis all types of RNA polymerases(I, II, III) for the synthesis of rRNA, tRNA, and mRNA No Transcription factor are Transcription factors (TFs) are involved involved TATA and TTGACA boxes are TATA, CAAT (CCAATC) and typically present GC (GGGCGG) boxes are present. Termination of transcription is Termination depends upon Rho dependent or special sequence on the independent transcript  The differences between replication and transcription Transcription Replication Definition Synthesis of RNA Synthesis of DNA Selective to one gene in All genes (the entire chromosome Gene code eukaryotes or a group of genes in prokaryotes RNA polymerase DNA polymerase Polymerase No primer is needed Need a primer Primer. Properties of polymerase Polymerase has no proofreading Polymerase has proofreading activity, activity, no exonuclease or exonuclease ,endonuclease activities ,endonuclease activities and no repair and repair the mistakes the mistakes Post synthesis modification There is post transcriptional Only methylation modifications