Transcription Lecture: A Comprehensive Overview - PDF

TRANSCRIPTION By Dr. Mohamed Gad  Definition  Transcription is defined as the synthesis of RNA from DNA,  Cellular RNAs include: 1. Messenger RNA (mRNA) 2. Ribosomal RNA (RNA) 3. Transfer RNA (IRNA) 4. Several small RNAs (1-2% of total RNA in the cell....

TRANSCRIPTION By Dr. Mohamed Gad  Definition  Transcription is defined as the synthesis of RNA from DNA,  Cellular RNAs include: 1. Messenger RNA (mRNA) 2. Ribosomal RNA (RNA) 3. Transfer RNA (IRNA) 4. Several small RNAs (1-2% of total RNA in the cell. There are about 30 different varieties. They are very stable. Small Nuclear RNAs (SnRNAs) are a subgroup of small RNA. 5. Micro-RNA (miRNA). They alter the function of mRNA.  All are transcribed from DNA.  The first three RNAS are involved in protein synthesis and snRNA is involved in mRNA splicing.  Central Dogma of Molecular Biology The information available in the DNA is passed to messenger RNA, which is then used for synthesis of a particular protein. Some Terms related to transcription 1. Upstream: the 3' direction of the template strand 2. Downstream: the 5' direction of the template strand 3. Promoter: Nucleotide sequence upstream the transcription unit, where the RNA polymerase begins attachment to DNA 4. Terminator: Nucleotide sequence downstream the transcription unit, marking the end of transcription 5. Transcription unit: includes the promoter, transcription region and the terminator 6. Consensus sequence: a specific sequence of nucleotides that plays the same role in different locations  BASIC REQUIREMENTS FOR RNA TRANSCRIPTION 1. TEMPLATE 2. SUBSTRATE 3. ENZYME 4. Promoters 5. Initiation Factors  TEMPLATE A single strand of DNA acts as a template to direct the formation of complementary RNA transcript. The strand that is transcribed to RNA molecule is referred to as the template strand and The mRNA base sequence is complementary the other DNA strand is to that of the template strand and identical referred as the coding strand. to that of the coding strand.  SUBSTRATE The substrates for RNA synthesis are the four ribonucleoside triphosphates: 1. ATP 2. GTP 3. CTP 4. UTP  ENZYME DNA dependent RNA polymerase, called RNA polymerase (RNAP), is responsible for the synthesis of RNA, 5' to 3' direction, using DNA template.  Prokaryotic RNA polymerase Prokaryotes have single RNA polymerase that transcribes all three RNAs. RNAP contains four subunits (2 α, β' β) which form the core enzyme. The active enzyme, the holoenzyme contains core enzyme and a fifth subunit called sigma (σ) factor. The σ factor is required for binding of the polymerase to specific promoter regions of DNA template. RNAP is a metalloenzyme containing Zn molecule  Eukaryotic RNA polymerase Eukaryotic cells have three RNA polymerases 1. RNA polymerase I: 18 S, 5.8 S & 28 S rRNA (Nucleolus) 2. RNA polymerase II: mRNA precursor & small RNAs 3. RNA polymerase III: tRNA & 5 S rRNA  Promoters Promoters are characteristic sequences of DNA which are different in prokaryotes and eukaryotes. Promoter sequences are responsible for directing RNA polymerase to initiate transcription at a particular point known as start point or initiation site. A base in the promoter region is assigned a negative number if it occurs prior, located in front (upstream) of the gene that is to be transcribed Position +1 indicates the first nucleotide that will be transcribed into RNA Sequences following the first base are numbered positively and are said to be downstream of the initiation point  Prokaryotic promoters  Prokaryotic genes have two promoter sequences: 1. Pribnow box (–10 region) has the nucleotide sequence TATAAT and is usually found 10 base pairs away from (upstream) the start point. 2. The –35 region, has the nucleotide sequence TTGACA. It is named – 35 sequence because it is found 35 base pairs away from (upstream) the start point.  Eukaryotic promoters  The promoters used by RNA polymerase I and II are similar to the prokaryotic promoter in that they are upstream of the start point  However, the promoters used by RNA polymerase III are unique because they are usually downstream of the start point  Examples: 1. Hogness box or TATA box: It is a stretch of six nucleotides and located 25 nucleotides upstream of the transcription starting point. 2. CAAT box: It is stretch of eight nucleotides and located about 75 nucleotides upstream of the transcription starting point. 3. GC box: It is a stretch of six nucleotides and is located about 90 nucleotides upstream of the transcription starting point.  Transcription of eukaryotic genes is further stimulated by enhancer sequences, which can be quite distant from the start site on either its 5' or its 3' side Prokaryotic & Eukaryotic promoters  Initiation Factors Initiation factors are needed to initiate transcription. In prokaryotes only a single factor namely sigma (σ) is needed to initiate transcription which enables the RNA polymerase holoenzyme to recognize and bind tightly to the promoter sequences. In eukaryotes multiple factors are required because of the diversity of promoters and complexity of their RNA polymerases  Stages of Transcription in prokaryotes 1. INITIATION 2. ELONGATION 3. TERMINATION  INITIATION  Initiation of transcription involves 1. binding of RNA holopolymerase (core enzyme + factor σ) to the template at the promoter site. 2. The binding of the RNA polymerase to the DNA template results in the unwinding of the DNA double helix. 3. The enzyme then catalyzes the formation of phosphodiester bond between the first two ribonucleotides complementary to DNA template sequence. 4. The first base is usually a purine.  Unlike the initiation of replication, transcriptional initiation does not require a primer  ELONGATION  Elongation proceeds after the formation of the first phosphodiester bond.  After formation of approximately 10 phosphodiester bonds of the new RNA, sigma (σ) subunit dissociates from the core enzyme.  The core enzyme then continues the elongation of the transcript.  The process of elongation of the RNA chain continues until a termination signal is reached.  TERMINATION In prokaryotes termination of transcription occurs by one of the two well characterized mechanisms: 1. Rho-dependent 2. Rho-independent.  Rho-dependent termination Rho-dependent termination, requires a protein factor called rho (ρ) which recognizes the termination signal and has an ATP dependent helicase activity that displaces the RNA polymerase from template resulting in termination of RNA synthesis.  Rho-independent termination Rho-independent termination brought about by the formation of a secondary structure (hair-pin loop) in the newly synthesized RNA, which removes the RNA polymerase from DNA template, resulting in the release of the transcript. This hairpin loop structure is followed by a sequence of four or more uracil residues. TRANSCRIPTION  DIFFERENCE BETWEEN PROKARYOTIC AND EUKARYOTIC TRANSCRIPTION Prokaryotes Eukaryotes Site Transcription and translation occur within the same Transcription and translation occur in different cellular cellular compartment. In fact, translation of bacterial compartments; Transcription occurs within the nucleus and mRNA begins during transcription translation outside the nucleus. Hence, translation can occur only after transcription has finished. Post Prokaryotes translate primary transcript of mRNA without Eukaryotes extensively process mRNA primary transcripts prior to transcription undergoing processing translation. process Promoters Promoter sites generally have the base sequence TATAAT Promoter sites generally have the base sequence TATAAA centered (pribnow box) centered at -10 and -35 region with base at base -25 or CAAT centered at -75 or GC box centered at -90 sequences of TGTTGACA. RNA Single kind of polymerase for all RNAs Three types of RNA polymerase. polymerase  INHIBITORS OF RNA SYNTHESIS  Rifampin It is an antituberculosis drug It inhibits the initiation of transcription by binding β-subunit of prokaryotic RNA polymerase. Rifampin has no effect on eukaryotic nuclear RNA polymerases  Dactinomycin (actinomycin D) Dactinomycin is a therapeutic agent in the treatment of some cancer. It binds tightly and specifically to double helical DNA and thereby prevents it from being an effective template for RNA synthesis At low concentrations, dactinomycin inhibits transcription without affecting DNA replication or translation. Hence, dactinomycin is extensively used as a inhibitor of the formation of new RNA in both prokaryotes and eukaryotes.  α-Amanitin it is a compound derived from the poisonous mushroom Amanita phalloides inhibits eukaryotic RNA polymerase.  ROLE OF ENHANCERS AND SILENCERS IN EUKARYOTIC TRANSCRIPTION  Enhancers are DNA sequences that regulate the frequency of transcription of genes in eukaryotic cells.  Enhancers are type of cis-acting element. A particular enhancer is effective only in certain cells, eg the immunoglobulin enhancers functions in B- lymphocytes but not elsewhere.  These DNA sequences have no promoter activity of their own but can stimulate the transcription of genes.  They differ from promoters in that 1. Their sequences are dissimilar 2. May be located thousands of base pairs from the start point of transcription. 3. They can be upstream, downstream, or within genes. 4. Enhancer are effective when present on either DNA strand.  SIMILARITY BETWEEN REPLICATION AND TRANSCRIPTION 1. They involve the general steps initiation, elongation and termination 2. Synthesis occurs in the 5' 3' direction 3. Follows Watson-Crick base pairing rules  DIFFERENCE BETWEEN REPLICATION AND TRANSCRIPTION TRANSCRIPTION REPLICATION Nucleotides Ribonucleotides Deoxyribonucleotides Bases Adenine, Guanine Adenine, Guanine, Cytosine & Uracil Cytosine & thymine Primer Not Required Required Size Only a very small portion of the genome is The entire genome must be copied during DNA transcribed into RNA replication Proofreading No proofreading Proofreading