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BIOL 130 Study Notes Unit V Gene Expression

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University of Waterloo
BIOL 130
Richard Ennis

BIOL 130 Unit V Study Notes Part I: DNA to RNA ­ Transcription  Central Dogma: • All cells seemingly follow the same central dogma, that is o DNA replication occurs; this is the information storage stage o RNA synthesis to create mRNA (transcription); this is the information carrier stage o Protein translation; this is the active cell machinery stage  Proteins are ultimately created from binding amino acids • The genotype is the gene sequence of the organism • When the central dogma occurs, that genotype determines the activity and function of a protein • The effect and activity of the protein directly affects the phenotype of the organism o This phenotype is the visible characteristic expressed in a cell as a result of the genotype RNA vs. DNA • RNA contains ribose as its sugar, which is best characterized by the 3 –OH group o This makes RNA less stable than DNA • DNA contains deoxyribose as its sugar • RNA uses base U (uracil) instead of base T (thymine) used by DNA • RNA is also more complex than DNA and is therefore able to form more complex folds Modifications to the Central Dogma: • There are many genes that code for different RNA molecules that are not necessarily directly related to the coding of proteins o mRNA is therefore not the only type of RNA molecule that genes can code for! o These other RNA molecules are involved in:  Regulating gene transcription  Processing mRNA prior to translation  Transport of amino acids during translation  Catalyzing peptide bond formations during translation • Types of RNA and their functions: o mRNA – codes for proteins (translation) o rRNAs – forms the core of ribosomes and catalyzes RNA synthesis o miRNAs – regulate gene expression o tRNAs – adaptors between mRNA and amino acids for protein synthesis o Other small RNAs – RNA splicing, telomere maintenance, etc. Transcription from a DNA Template: • Only one strand of DNA is transcribed; this is known as the template strand o This template strand is used by RNA polymerase to synthesize RNA o RNA becomes complimentary to the template strand when it uses it as a template • The other DNA strand is the non-template (coding) strand o This DNA strand matches with RNA, except RNA uses a uracil base instead of a thymine base. • RNA is: o complimentary to template strand and o matches the non-template strand Transcription in Prokaryotes: • Prokaryotic RNA polymerase (RNApol) o Large, globular enzyme with channels running through it with an active site at where the channels intersect o RNA polymerase transcribes DNA and makes RNA • Machinery used in transcription: o The sigma factor:  Most bacteria have several types of sigma proteins  These sigma factors recognize a promoter sequence and binds to these promoters  This tells RNA where to begin transcription o Holoenzyme (a type of RNA polymerase) is made up of a core enzyme  Has the ability to synthesize RNA and the regulatory subunit (sigma factor)  Transcription of Prokaryotes: INITIATION • Transcription is initiated at specific sections of DNA called the promoter regions o Promoters have 2 key regions  -10 box (i.e. 10 bases upstream from the start site)  -35 box (i.e. 35 bases upstream; transcript starts at +1) • Once the sigma factor identities and binds to the promoter regions at the -10 box and -35 box, RNA polymerase holoenzyme properly orients and begins transcription at the start site  Transcription in Prokaryotes: ELONGATION • Because RNA is complimentary to the template strand, the template strand is the only strand that is transcribed by the holoenzyme • Therefore, when the DNA double helix is fed into RNA polymerase holoenzyme, the sigma factor will open the helix to begin transcription • The template strand goes to the active site, whereas the non-template strand goes away from the active site • RNA is synthesized using the template strand to make itself complimentary to the template strand • The sigma factor will release, but transcription will still continue to elongate the growing RNA strand • The growing RNA strand can exit through another site Transcription in Prokaryotes: TERMINATION • RNA polymerase holoenzyme will stop transcription when it reaches a termination signal in the DNA template • The RNA folds back on itself to form a hairpin structure o This hairpin structure disrupts the transcription complex causing RNA polymerase to separate from the completed RNA chain Fun fact: Prokaryotes are capable of concurrent transcription translation! Transcription in Eukaryotic Cells: • Unlike prokaryotic cells, which carry out transcription in the cytoplasm, eukaryotes carry out transcription in the nucleus. • Key differences between their transcription processes will be addressed later on Transcription in Eukaryotes: INITIATION • RNA polymerase II will only bind to the promoter region if the transcription factors have already bound to the promoter region. o Transcription factors are accessory proteins that help mediate RNA polymerase II and they also create the transcription initiation complex when combined with RNA polymerase II • If the transcription factors have bound on, then RNA polymerase binds to the promoter region which includes the transcription start point; o This region is identified by the sequence TATA about 25 nucleotides upstream from the start point. • DNA will then be unwound and transcription will begin Transcription in Eukaryotes: Elongation • RNA polymerase II moves from 5’  3’ and unwinds the DNA to build the RNA transcript, and then rewinds what has been transcribed o It uses the template strand and adds the appropriate complimentary ribonucleotides to the growing RNA transcript • A single gene can be transcribed simultaneously by several molecules of RNA polymerase at the same time Transcription in Eukaryotes: Termination • Eventually RNA polymerase will transcribe the terminator sequence, which signals the end of transcription • This causes the RNA polymerase to detach from the RNA transcript (which is the actual mRNA) • The mRNA then has its introns removed (RNA splicing), is capped at the 5’ end, and the addition of a poly(A) tail is added to the 3’ (polyadenylation). o Only once all of this has occurred, the “mature” mRNA is released out of the nucleus to go to the translation process. Fun Fact: Unlike prokaryotes, eukaryotes cannot synthesize RNA and translate it at the same time. Eukaryotic Transcription vs. Prokaryotic Transcription: 1. Eukaryotes need to deal with DNA packaging since the DNA is wrapped around histones and is tightly packed 2. Eukaryotes have 3 distinct types of RNA polymerase (RNA polymerase I, II, and III), which are each responsible for synthesizing different types of RNA molecules (not just mRNA); prokaryotes only have 1 type of RNA polymerase (holoenzyme) o RNA polymerase I – transcribes most rRNA genes o RNA polymerase II – transcribes protein-coding genes, miRNA genes, and genes for some small RNAs (e.g. those in spliceosomes) o RNA polymerase III – tRNA genes, 5S rRNA genes, and genes for other small RNAs o Remember RNA polymerase I = rRNA; RNA polymerase II = mRNA, genes for spliceosomes (both of which have to do with transcription), and miRNA; RNA polymerase III = tRNA 3. Eukaryotic RNA polymerase II requires help from other accessory proteins o These are called transcription factors that assemble at the promoter region with RNA polymerase II i. TBP transcription factor recognizes the promoter region TATA ii. TBP + TFIID distorts the double helix to allow other factors to pile on to form a “transcription initiation complex” o Thus, eukaryotic RNA polymerase II will only bind onto the promoter region when certain transcription factors have already bound on. 4. Eukaryotes only send out mature mRNA from nucleus after processing (i.e. 5' capping and 3’ polyadenylation) o The mRNA is capped at its 5’ end, which allows the machinery in the later translation process to recognize it o Polyadenylation occurs near the 3’ end where a poly(A) tail is added to protect from degradation o Exon junction complex binds to properly spliced mRNA and only then can it be transported out 5. Eukaryotic genes can be spread out, with gaps of untranscribed DNA between them, which allows for complex regulation of gene transcription by regulatory sequences in the genome o Coding regions are called exons; these sequences are interrupted by gaps of non-coding regions called introns: o Functional mRNA is made by transcribing the entire gene (including the exons AND introns) o But after the capping of the 5’ end, RNA splicing will occur during transcription to remove the introns i. RNA splicing  carried out by spliceosomes  Each spliceomere has 5 small nuclear ribonucleoproteins (snRNPs) ii. Spliceosome activity is catalyzed by ribozymes Intron Removal Process: RNA Splicing • There are special sequences that indicate the beginnings and endings of noncoding intron regions o These are borders between exons and an adjacent introns • These special sequences are recognized the snRNPs of the spliceosome, allowing them to locate and cleave at intron-exon borders • There is a point known as “branch point A (adenine)” that attacks the 5’ splice site, cutting the sugar-phosphate backbone o The cut end forms covalent bonds with ribose sug
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