Lecture 2 Replication and Transcription PDF

DNA Replication and Transcription Ass.Prof./ Dalia Shaalan Department of Medical Biochemistry and Molecular Biology Contents: Definition of DNA replication. Requirements and steps of eukaryotic DNA replication. Post replication modification of DNA. Definition of transcript...

DNA Replication and Transcription Ass.Prof./ Dalia Shaalan Department of Medical Biochemistry and Molecular Biology Contents: Definition of DNA replication. Requirements and steps of eukaryotic DNA replication. Post replication modification of DNA. Definition of transcription. Requirements and steps of eukaryotic transcription Post transcription modification of RNA (Seminar 3 Discussion). Learning outcomes: Identify DNA replication. List requirements needed for eukaryotic DNA replication Recognize the steps of eukaryotic DNA replication Identify transcription. List enzymes & proteins needed for eukaryotic transcription Recognize the steps of eukaryotic transcription Discuss post-transcriptional modifications of RNA (Seminar 3).  Replication It is the duplication of the DNA content before mitosis. DNA replication proceeds through multiple enzymatic activities done by DNA polymerases Complex. Semi-conservative replication of DNA Replication of DNA occurs in semi- conservative manner. When the two strands of DNA are separated each one acts as a template for the replication of a new daughter complementary strand. Each new DNA molecule contains one of the original strands and a new complementary strand.  Requirements for DNA replication The template DNA strands. Deoxyribonucleotides triphosphate (dNTP; dATP, dGTP, dCTP and dTTP). A primer to build new DNA strand upon it. DNA Polymerases Complex. Replication proteins and enzymes.  Steps of DNA Replication I. Separation of the two DNA strand. 1. Initiation = Identification of the origin of replication. 2. Unwinding of DNA=formation of replication fork. 3. Stabilization of the fork. II. DNA synthesis by polymerase. 1. Priming by primase enzyme. 2. Synthesis of new DNA strands. III. Termination.  I. Separation of the two DNA strand. 1- Initiation = Identification of the origin of replication. DNA replication start at multiple sites (in eukaryotes) called origin of replication or (ori). Ori Consists of unique sequence of A-T bases that is recognized by the enzymes and proteins responsible for initiation of replication. I. Separation of the two DNA strand (cont.) 2. Unwinding of DNA=formation of replication fork. DNA helicase unwind the DNA at the origin of replication onwards to generate a "replication bubble“ forming two V-shaped replication forks. I. Separation of the two DNA strand (cont.) 3. Stabilization of the fork: The two strands are kept unpaired by Pre-priming complex proteins (both DNA helicase and Single- stranded DNA binding proteins (SSBPs)). SSBPs protect the single stranded template from nucleases. SSBPs are easily removed by the DNA polymerase enzyme.  II. DNA synthesis by polymerases. Each DNA strand will act as a template to direct the synthesis of a new daughter DNA strand. This occur by DNA polymerases complex. DNA polymerases cannot initiate DNA synthesis by themselves. A primer is needed.  DNA Polymerases Complex In eukaryotes e.g., human cell, there are 5 components of DNA polymerases complex α, β, γ, δ and ε. The complex can copy the template strand in the 3’ to 5’ direction and synthesis the new strand in the 5’ to 3’direction. It can not initiate DNA synthesis ; it needs a primer to build new DNA strand upon it. It can proofread the synthesized strand and identify the copying errors to correct them.  II. DNA synthesis by polymerases. DNA polymerases enzyme synthesize DNA in :- – Continuous coping of parental strand (in the direction from Ori towards replication fork= 3’ to 5’ ) → leading strand (5’ to 3’ towards the fork). – Discontinuous coping of parental strand (in the direction from the replication fork away to the Ori = 3’ to 5’) → Lagging strand (5’ to 3’ away from the fork).  II. DNA synthesis by polymerase. 1. Priming by primase enzyme: The primase (a type of RNA polymerase) synthesizes a short segment of RNA (7-10 bases) as a primer utilizes the parent DNA strands as templates. The primer is a starting sequence for the DNA polymerase to add nucleotide upon and proceed the DNA replication. II. DNA synthesis by polymerase (cont.). 1. Priming by primase enzyme (cont.): One RNA primer is formed for the leading strand and multiple primers for the lagging strand. RNA primer is in 5`→3` direction according to base pairing rule A-U and G-C using 3`→5` of DNA strand as template. RNA primer with a free hydroxyl group on the 3′-end of the RNA strand, act as acceptor of DNA nucleotide by DNA polymerase. II. DNA synthesis by polymerase (cont.). 2. Synthesis of leading strand: The DNA polymerases bind to the RNA primer. They can read the parental DNA from 3` →5` and synthesize a newly formed DNA strand in the 5`→3` direction. Synthesis of DNA proceeds through attachment of the 5’ phosphate of incoming nucleotide (dNTP) to the existing 3’ OH of RNA primer with release of pyrophosphate (ppi).  II. DNA synthesis by polymerase (cont.). 2. Synthesis of lagging strand: Replicated in a direction away from the replication fork in the form of small fragments called Okazaki fragment because the DNA polymerase always synthesize in the 5`→3` direction of the new DNA.  II. DNA synthesis by polymerase (cont.). 2. Synthesis of lagging strand: Each Okazaki fragment is a small DNA sequence (100 to 200 nucleotides) connected to one RNA primer. After many Okasaki fragments are formed, all RNA primers will be removed, and the space left will be filled by DNA polymerase. Then all fragments will be joined together by DNA ligase. Removal of RNA primer and filling of the “gaps” by DNAP  III. Termination. First, polymerase enzymes proofread the sequences to ensure correct nucleotides pairing. Then, correcting errors to remove the incorrect DNA and fill the gap. Lastly, recoiling (twisting) of the parent and newly synthesized strand will result in completion of the replication process.  Post-Replication Modification of DNA The major post-replication modification of DNA is methylation. DNA methylation involves the addition of a methyl group to the 5th position of the cytosine pyrimidine ring. This modification can be inherited through cell division.  Question 1 In replication, separation of the double strands of DNA is catalyzed by: A. Helicase. B. Primase. C. DNA polymerase. D. RNA polymerase. E. Ligase.  Question 2 DNA replication requires all the following EXCEPT: A. DNA polymerase B. A short RNA primer. C. Ribonucleoside triphosphates. D. DNA template E. DNA ligase  Flow of genetic information (Central dogma) In almost all organisms , the flow of genetic information goes in one direction from the stored gene code in DNA by transcription into a message copy as mRNA that is then translated into a protein that carryout the gene function.  Flow of genetic information (Central dogma) The mechanism of flow of genetic information is reversed in Retroviruses (RNA viruses ) i.e. from RNA into DNA then into mRNA then protein during the life cycle of these viruses.  Transcription It is RNA synthesis from a DNA template strand catalyzed by RNA polymerase.  Transcription Any strand of DNA may serve as a template (anticodon strand) for transcription.  Required materials for Transcription 1. Transcription unit (DNA template: promoter region, transcribed region, termination region and regulatory elements). 2. Four ribonucleotide triphosphate (ATP, GTP, UTP and CTP). 3. RNA polymerase enzymes. 4. Transcription factors.  Transcription unit structure 1. Promoter region (DNA sequences upstream of transcription start site): It is DNA sequence located at the beginning of the gene. Important for initiation of transcription to be recognized by RNA polymerase and initiation factors.  Transcription unit structure (cont.) Eukaryotic promoter is composed of : Hogness or (TATA box) located 25 bases before starting point of transcription. CAAT box located about 70 to 80 bases before transcription starting point. Sometimes GC boxes  Transcription unit structure (Cont.) 2. Transcription region: It is a sequence of nucleotide to be transcribed into RNA molecules. It is composed of Exons interrupted by Introns. The exons are coding sequences of the gene, and the introns are non-coding sequences. 3. Termination region: is the sequence of DNA located at the end of transcribed region. Transcription unit structure (Cont.) 4. Transcription regulatory elements : They are DNA sequences located either upstream, downstream the gene or even within gene. They regulates the rate of transcription. They include: Enhancers which increase the transcription rates. Silencers which decrease the transcription rates.  RNA Polymerase Enzyme (RNAP) RNA polymerase reads the template strand (anticodon strand) in the 3’ to 5’ direction to synthesize a single stranded RNA in the 5’ to 3’ direction. It utilizes ribonucleotide triphosphates (NTP) as building units. It can initiate RNA synthesis without the need of a primer.  RNA Polymerase Enzyme (RNAP) Types of RNA polymerase: Type Transcribe RNA polymerase I rRNA (28S, 18S, 5.8S). RNA polymerase II mRNA and most small nuclear RNA (snRNA). RNA polymerase III tRNA, some snRNAs, and 5S rRNA. Steps of transcription (1) Initiation. (2) Elongation. (3) Termination. After attachment of RNAP at the promoter, transcription begins at the “start point” and synthesis of RNA continues (elongation) until a termination sequence (terminator) is reached.  1- Initiation In eukaryotes: multiple transcription factors bind the promoter with RNA polymerase to initiate transcription. Binding of initiation factors and RNAP to DNA template at the promoter leads to local unwinding of the DNA double helix. The enzyme then catalyze the formation of phospho-diester bond between the first two bases. RNAP does not require a primer.  2- Elongation - RNAP begins to synthesize a transcript at the 5’ end (usually starting with a purine). - RNAP uses ribonucleotides triphosphates (ATP, GTP,UTP,CTP) according to the base pairing rule, and releases ppi each time.  2- Elongation (cont.) Elongation of RNA chain continues until a termination region reached.  3- Termination In eukaryotes, termination started when RNAP meets the terminator region. RNAP releases 10-35 base pairs after termination signal.  Question 3 To start transcription, RNA polymerase should recognize the: A. Transcribed region. B. Enhancer sequence. C. Silencer sequence. D. Promotor region. E. Regulatory region.  Question 4 According to the given DNA template: 5’-GTC ACC CCG TTA GCT CGT CAA GTC TCA-3’ 1. Construct the complementary DNA sequence. 2. Predict the transcribed mRNA sequence. Summary References