TRANSCRIPTION.pdf

TRANSCRIPTION  Incident: On November 8, 2009, Tomasa, 31, consumed wild mushrooms mistaken for edible ones, leading to severe poisoning. Symptoms appeared within hours; Tomasa died of liver failure after three weeks. Mushrooms: Amanita phalloides (Death Cap)...

TRANSCRIPTION  Incident: On November 8, 2009, Tomasa, 31, consumed wild mushrooms mistaken for edible ones, leading to severe poisoning. Symptoms appeared within hours; Tomasa died of liver failure after three weeks. Mushrooms: Amanita phalloides (Death Cap) A single death cap can kill an adult. Death rate: 22% overall; >50% in children under 10.  Death Cap Poisoning Symptoms: Initial: Abdominal pain, cramping, vomiting, diarrhea (6-12 hours after consumption) Remission phase often misleading Severe: Liver cell death, permanent damage, potential death within days. Toxin: α-Amanitin Inhibits RNA polymerase II Stops RNA and protein synthesis Results in cell death, particularly in the liver  Transcription of an RNA Molecule from a DNA Template Transcription synthesizes RNA from DNA templates. Transcription is selective, transcribing only necessary genes. 3 major components required : DNA Template: The blueprint for RNA synthesis. Raw Materials: Substrates needed to build a new RNA molecule. Transcription Apparatus: Proteins required for RNA synthesis.  The Transcribed Strand Transcription uses a single strand of the DNA double helix. Only one strand of DNA, called the template strand, is transcribed. Template Strand: Used for RNA synthesis; RNA is complementary and antiparallel to this strand. Nontemplate Strand: Not ordinarily transcribed; has the same polarity and base sequence as RNA, but with T instead of U. Different genes may be transcribed from different DNA strands.  Transcription Unit Sequence of nucleotides in DNA that encodes a single RNA molecule and the sequences necessary for its transcription. Key Components: Promoter RNA-Coding Region Terminator  Promoter: DNA sequence where the transcription apparatus binds.Indicates the template strand and direction of transcription. Determines the transcription start site. RNA-Coding Region: Sequence of DNA nucleotides copied into an RNA molecule. Terminator: Sequence of DNA nucleotides signaling the end of transcription. Part of the RNA-coding sequence; transcription stops after copying the terminator.  Substrate for Transcription RNA synthesized from ribonucleoside triphosphates (rNTPs). Two phosphate groups are cleaved from the incoming rNTP. The remaining phosphate forms a phosphodiester bond with the RNA molecule.  Bacterial RNA Polymerase Catalyzes the synthesis of all classes of bacterial RNA: mRNA, tRNA, and rRNA. Large multimeric enzyme with several polypeptide chains. Core enzyme consists of five subunits: Two alpha (α) One beta (β) One beta prime (β′) One omega (ω) (stabilizes the enzyme, not essential for transcription)  Bacterial RNA Polymerase Core enzyme is responsible for elongation of the RNA molecule by adding RNA nucleotides. Sigma (σ) factor controls the binding of RNA polymerase to the promoter. Forms a holoenzyme when associated with the core enzyme. Ensures stable binding to the promoter and proper initiation of transcription. Detaches after the initiation of RNA synthesis. Bacteria may have various sigma factors, each directing RNA polymerase to different sets of promoters.  Rifamycins and RNA Polymerase Inhibition Rifamycins: A group of antibiotics that inhibit RNA polymerase. Widely used to treat tuberculosis, which kills nearly 2 million people annually. Binds to bacterial RNA polymerase, jamming the part that clamps onto DNA. Prevents RNA polymerase from interacting with the DNA promoter. Inhibits bacterial RNA polymerases without affecting eukaryotic RNA polymerases due to structural differences.  EUKARYOTIC RNA POLYMERASES Most eukaryotic cells possess three distinct types of RNA polymerase, each of which is responsible for transcribing a different class of RNA. All eukaryotic polymerases are large multimeric enzymes, typically consisting of more than a dozen subunits. Some subunits are common to all RNA polymerases, whereas others are limited to one of the polymerases. As in bacterial cells, a number of accessory proteins bind to the core enzyme and affect its function What is the function of the sigma factor?  BACTERIAL TRANSCRIPTION Transcription in bacteria involves three main stages: Initiation: The transcription apparatus assembles on the promoter region of the DNA. RNA polymerase binds to the promoter and starts RNA synthesis. Elongation: The DNA is threaded through the RNA polymerase. RNA polymerase unwinds the DNA and adds nucleotides to the 3′ end of the growing RNA strand. Termination: RNA polymerase recognizes the end of the transcription unit and the RNA molecule is released from the DNA template.  Transcription Initiation Step 1: Promoter Recognition Step 2: Formation of Transcription Bubble Step 3: Creation of First Bonds between rNTPs Step 4: Escape of Transcription Apparatus from Promoter  PROMOTER BINDING Selectivity of transcription enforced through promoter binding Determines which DNA template parts are transcribed and how often Different genes transcribed with different frequencies Promoter binding determines transcription frequency Promoters have varying affinities for RNA polymerase Affinity can change over time due to interactions and other factors  Bacterial Promoters Function: Define the transcription start site, which DNA strand to read, and the direction of RNA polymerase movement. Location: Usually adjacent to the RNA-coding region. Consensus Sequences: Short, conserved nucleotide stretches found in many bacterial promoters, indicating functionally significant regions essential for RNA polymerase binding and transcription initiation.  –10 Consensus Sequence (Pribnow Box) Key feature in bacterial promoters, located approximately 10 bp upstream of the transcription start site. TATAAT TATAAT is just the consensus sequence—representing the most commonly encountered nucleotides at each of these sites In most prokaryotic promoters, the actual sequence is not TATAAT  –35 consensus sequence Another consensus sequence common to most bacterial promoters is TTGACA, which lies approximately 35 nucleotides upstream of the start site. Is termed the –35 consensus sequence The nucleotides on either side of the -10 and -35 consensus sequences and those between them vary greatly from promoter to promoter, suggesting that these nucleotides are not very important in promoter recognition In bacterial promoters, consensus sequences are found upstream of the start site, approximately at positions -10 and -35.  Elongation At the end of initiation, RNA polymerase undergoes a change in conformation (shape) and thereafter is no longer able to bind to the consensus sequences in the promoter. This change allows the polymerase to escape from the promoter and begin transcribing downstream. The sigma subunit is usually released after initiation, although some populations of RNA polymerase may retain sigma throughout elongation.  As it moves downstream along the template, RNA polymerase progressively unwinds the DNA at the leading (downstream) edge of the transcription bubble Joining nucleotides to the RNA molecule according to the sequence on the template, and rewinds the DNA at the trailing (upstream) edge of the bubble. In bacterial cells at 37°C, about 40 nucleotides are added per second. This rate of RNA synthesis is much lower than that of DNA synthesis, which is 1000 to 2000 nucleotides per second in bacterial cells.  The transcription bubble Transcription takes place within a short stretch of about 18 nucleotides of unwound DNA—the transcription bubble. Within this region, RNA is continuously synthesized, with single-stranded DNA used as a template. About 8 nucleotides of newly synthesized RNA are paired with the DNA- template nucleotides at any one time. As the transcription apparatus moves down the DNA template, it generates positive supercoiling ahead of the transcription bubble and negative supercoiling behind it. Topoisomerase enzymes probably relieve the stress associated with the unwinding and rewinding of DNA in transcription, as they do in DNA replication  Accuracy of transcription Although RNA polymerase is quite accurate in incorporating nucleotides into the growing RNA chain, errors do occasionally arise. When RNA polymerase incorporates a nucleotide that does not match the DNA template, it backs up and cleaves the last two nucleotides (including the misincorporated nucleotide) from the growing RNA chain. RNA polymerase then proceeds forward, transcribing the DNA template again.  Termination RNA polymerase adds nucleotides to the 3′ end of the growing RNA molecule until it transcribes a terminator. At the terminator, several overlapping events are needed to bring an end to transcription: RNA polymerase must stop synthesizing RNA The RNA molecule must be released from RNA polymerase The newly made RNA molecule must dissociate fully from the DNA RNA polymerase must detach from the DNA template.  Termination Bacterial cells possess two major types of terminators. Rho-dependent terminators are able to cause the termination of transcription only in the presence of an ancillary protein called the rho factor. Rho-independent terminators (also known as intrinsic terminators) are able to cause the end of transcription in the absence of rho.  Rho-independent terminators Rho-independent terminators, which make up about 50 percent of all terminators in prokaryotes, have two common features. First, they contain inverted repeats (sequences of nucleotides on one strand that are inverted and complementary). When inverted repeats have been transcribed into RNA, a hairpin secondary structure forms.  Second, in rho-independent terminators, a string of seven to nine adenine nucleotides follows the second inverted repeat in the template DNA. Their transcription produces a string of uracil nucleotides after the hairpin in the transcribed RNA.  The string of uracils in the RNA molecule causes the RNA polymerase to pause, allowing time for the hairpin structure to form. Evidence suggests that the formation of the hairpin destablizes the DNA–RNA pairing, causing the RNA molecule to separate from its DNA template. Separation may be facilitated by the adenine–uracil base pairings, which are relatively weak compared with other types of base pairings. When the RNA transcript has separated from the template, RNA synthesis can no longer continue.  Rho-dependent terminators Rho-dependent terminators have two features: (1) DNA sequences that produce a pause in transcription (2) a DNA sequence that encodes a stretch of RNA upstream of the terminator that is devoid of any secondary structures. This unstructured RNA serves as a binding site for the rho protein, which binds the RNA and moves toward its 3′ end, following the RNA polymerase. When RNA polymerase encounters the terminator, it pauses, allowing rho to catch up. The rho protein has helicase activity, which it uses to unwind the RNA–DNA hybrid in the transcription bubble, bringing transcription to an end.  Polycistronic mRNA In bacteria, a group of genes is often transcribed into a single RNA molecule, which is termed a polycistronic RNA. Thus, polycistronic RNA is produced when a single terminator is present at the end of a group of several genes that are transcribed together, instead of each gene having its own terminator. Typically, each eukaryotic gene is transcribed and terminated separately, and so polycistronic mRNA is uncommon in eukaryotes.  The Basic Rules of Transcription 1. Transcription is a selective process; only certain parts of the DNA are transcribed at any one time. 2. RNA is transcribed from single-stranded DNA. Within a gene, only one of the two DNA strands—the template strand—is normally copied into RNA. 3. Ribonucleoside triphosphates are used as the substrates in RNA synthesis. Two phosphate groups are cleaved from a ribonucleoside triphosphate, and the resulting nucleotide is joined to the 3′-OH group of the growing RNA strand. 4. RNA molecules are antiparallel and complementary to the DNA template strand. Transcription is always in the 5′→3′ direction, meaning that the RNA molecule grows at the 3′ end.  5. Transcription depends on RNA polymerase—a complex, multimeric enzyme. RNA polymerase consists of a core enzyme, which is capable of synthesizing RNA, and other subunits that may join transiently to perform additional functions. 6. A sigma factor enables the core enzyme of RNA polymerase to bind to a promoter and initiate transcription. 7. Promoters contain short sequences crucial in the binding of RNA polymerase to DNA; these consensus sequences are interspersed with nucleotides that play no known role in transcription. 8. RNA polymerase binds to DNA at a promoter, begins transcribing at the start site of the gene, and ends transcription after a terminator has been transcribed