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Chapter 16

Chapter 16 Transcription and Translation Notes.docx

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Lovaye Kajiura

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Chapter 16: Transcription and Translation DNA (Information Storage) Transcription mRNA (Information Carrier) Translation Protein (Product)  The first step of the synthesis of protein is the transcription of a gene and the production of messenger RNA.  The RNA message is a short lived copy of the archived instructions in DNA.  The sequence in ribonucleotides in the mRNA is then translated into amino acids. Transcription in Bacteria  RNA polymerases responsible for synthesizing mRNA.  Template directed synthesis 5’  3’  Transcription occurs when RNA pol matches the base ribonucleotide triphosphate with the complementary base in a gene- a section of DNA that codes for a protein or RNA.  Once a matching ribonucleotide is in place, RNA pol catalyzes the formation of a phosphodiester bond between the 3’ end of the growing mRNA chain and the new ribonucleotide.  Template strand= the strand being read by the enzyme.  Non-template strand= aka non coding strand because its sequence matches the sequence of the RNA that is transcribed from the template strand and codes for a polypeptide.  RNA has Uracil instead of Thymine in the coding strand. Non-template (coding) strand DNA RNA Template Strand Phosphodiester bond RNA Hydrogen Bonds DNA Template RNA polymerase  RNA pol is large and globular and has several prominent channels running through its anterior.  The enzymes active site is located where several of these channels intersect.  DNA fits into one of the enzyme’s channels and the two strands of the double helix separate inside the enzyme to expose a single – stranded template at the active site.  The enzyme can’t initiate transcription on its own.  The detachable protein subunit called sigma must bind to RNA pol before transcription can begin. RNA pol and sigma form a holoenzyme.  A holoenzyme consists of a core enzyme, which contains the active site for catalysis and other required proteins.  When researchers mixed RNA pol, sigma, and DNA together, they found that the holoenzyme bound tightly to specific sections of DNA, and these binding sites are known as promoters. Promoters are the sections of DNA where transcription begins.  Sigma’s function is regulatory; appears to be responsible for guiding RNA pol to specific locations where transcription should begin.  In bacteria, promoters located on the non-template strand and are 40-50 base pairs long.  Contains particular segments which look the same.  These segments are identical or similar to TATAAT, -10 box (called the -10 box because it is located 10 bases from the point where RNA pol starts transcription. It is located 10 bases upstream from the transcription start site.)  Contains TTGACA, -35 box  Follow up research shows that transcription begins when sigma binds to the -35 and -10 boxes. In other words, sigma makes initial contact with DNA and starts transcription, not RNA pol. INITIATION 1. Sigma binds to a promoter, and the DNA helix opens and creates two strands of single stranded DNA. 2. The template strand is threaded through a channel that leads to the active site inside RNA pol. 3. Ribonucleoside triphosphates (NTPs), enter a channel at the bottom of the enzyme and diffuse to the active site. 4. Incoming NTP pairs with a complementary base on the template strand of DNA, causing RNA pol to begin. This is an exergonic reaction caused by the RNA pol as well as a spontaneous reaction due to NTPs which have so much potential energy due to their 3 phosphate groups. 5. Sigma is released once RNA synthesis is underway. ELONGATION  RNA pol moves in the 3’  5’ direction of the template strand, synthesizing RNA in the 5’  3’ direction.  In the interior of the enzyme, a group of projecting amino acids called the enzyme’s zipper opens the double helix at the upstream end.  A nearby group of amino acids called the rudder helps steer the template and non- template strand through channels inside the enzyme.  The enzyme’s active site catalyzes the addition of nucleotides to the 3’ end of the growing RNA molecule at the rate of about 50 nucleotides per second.  During the elongation phase of transcription, all the prominent channels are filled. TERMINATION  In most cases, transcription stops when RNA pol reaches a stretch of DNA sequence that functions as a transcription termination signal.  As soon as the bases reach the stop sequence on the DNA, the RNA sequence folds back on itself and forms a short double helix that is held together by complementary base pairing.  The secondary structure that results is called a hairpin.  The formation of the hairpin structure is thought to disrupt the interaction between RNA pol and RNA transcript, causing a physical separation of the enzyme and product.  Basically: Transcription begins when sigma, as a part of the holoenzyme complex, binds to the promoter at the start of a gene. Once binding occurs, RNA pol begins to synthesize mRNA by adding ribonucleotides that are complementary to the template strand in DNA. Transcription ends when a termination signal at the end of the gene leads to the formation of a hairpin in the mRNA, disrupting the transcription complex. Transcription and RNA Processing in Eukaryotes  In eukaryotes, RNA pol does not bind directly to the promoter sequence by itself. Instead, proteins called basal transcription factors initiate eukaryotic transcription by matching the enzyme with the appropriate promoter region in DNA.  Differences between transcription in bacteria and eukaryotes: Bacteria Eukaryotes Sigma Basal or general transcription factors as well as activators and inhibitors TFs One Pol 3 Pols: - RNA pol II transcribes the genes that code for proteins (produces mRNA). - RNA pol I make the large RNA molecules found in ribosomes. - RNA pol III manufactures one of the small RNAs that are found in ribosomes as well as tRNA required for translation. -10 and -35 box More complex promotors TATA box, -30 bp No RNA processing RNA processing for mRNA that leaves the nucleus  To make functional mRNA in eukaryotes, certain sequences in genes must be disposed of and the separate coding sections are then combined to form a whole.  Philip Sharp carried out an experiment in the 1970’s to deter
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