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Chapter 8

BIOL 205 Chapter 8: BIOL205 Chapter 8 Textbook Notes

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BIOL 205
Ian Chin- Sang

Chapter 8 – RNA: Transcription and Processing - Genes of higher eukaryote are usually composed of exons that are coding, expressed regions, and introns, non-coding regions - A spliceosome removes the introns and joins the exons in a process called RNA splicing - Humans have about 21 000 genes that encode for 100 000 proteins because of alternative splicing - Non-protein-coding RNA (ncRNA) perform many essential roles - RNA copy of the gene must be synthesized in a process called transcription o First step is to copy, or transcribe, the information into a strand of RNA, using DNA as a template o Converted into an amino acid chain (protein) by translation - In eukaryotes, these take place in two different places, the former being in the nucleus and the latter being in the cytoplasm o Before going into the cytoplasm, undergoes removal of introns, 5’ cap and Poly-A tail addition - Two principles that this is based on 1. Complementarity of bases is responsible for determining sequence of RNA transcript in transcription 2. Certain proteins recognize particular base sequences in DNA and RNA – binding proteins bind to here and act on them 8.1 RNA Early Experiments Suggest and RNA Intermediate - Volkin and Astrachan found that when they infected E. coli with a T2 phage, a quick burst of RNA synthesis occurred, but “turned over” quickly o Brief lifetime, but suggested that it may be necessary in expression of T2 genome to make more virus particles - Pulse-chase experiment: infected bacteria are fed radioactive uracil (needed for only RNA synthesis so that it is labelled) and then after a while, rinsed and fed non- radioactive uracil o This “chases” the label out of RNA because radioactive ones break down and only the other ones are available o RNA analyzed shortly after “pulse” of radioactivity, is labelled, and other is not Properties of RNA 1. Ribose sugar in nucleotides rather than deoxyribose; RNA contains a hydroxyl group (OH) on the 2’ carbon atom and DNA only has an H 2. RNA is usually single-stranded nucleotide chain, not double stranded helix a. Much more flexible -> Intramolecular base pairing (with its own bases) 3. RNA molecules contain the pyrimidine base uracil (U) instead of thymine (T) a. Forms two hydrogen bonds with adenine just like T b. Capable of pairing with G (only during RNA folding, not transcription) c. U and G is weaker than U and A, but this versatility allows for complicated structures to be composed 4. RNA can catalyze biological reactions with ribozyme (RNA protein enzymes) Chapter 8 – RNA: Transcription and Processing Classes of RNA - Messenger RNA (mRNA): encodes information necessary to make proteins o Gene influences phenotype through gene expression o Only an immediate intermediate product - Functional RNA: does not encode information to make protein, but is the final functional product o Never translated into polypeptides** o Transfer of information from DNA to protein, processing of RNA, regulation of RNA and protein levels in the cell o Transfer RNA (tRNA) are responsible for bringing correct AA to the mRNA during translation o Ribosomal RNA (rRNA) major components of ribosomes that guide assembly of AA chain by mRNAs and tRNAs - Encoded by a small number of genes, but rRNAs account for a very large % of RNA in cell - Functional RNA Specific to Eukaryotes: o Small nuclear RNAs (snRNAs) further process RNA transcripts; some unite with several protein subunits to form spliceosome o MicroRNAs regulate the amount of protein produced by many eukaryotic genes o Small interfering RNAs (siRNAs) and piwi-interacting RNAs protect integrity of plant and animal genomes - Long noncoding RNAs (ncRNAs) transcribed from most regions of the genomes of humans and other animals and plants; function unknown - These RNAs are constitutive, so continually synthesized 8.2 Transcription - RNA produced by process that copies the nucleotide sequence of DNA: transcription o RNA is then called a transcript Overview: DNA as Transcription Template - Two strands of DNA separate and one acts as the template for RNA synthesis - In any one gene, only one strand is used and in that gene, it is always the same strand, starting at the 3’ end of template gene - Nucleotides form stable pairs with their complimentary bases in template o Placed by RNA polymerase linking ribonucleotides and making an ever-growing strand of RNA - RNA grows in 5’ to 3’ direction (always added to the 3’ end) o Because nucleotides are oppositely oriented, this means that the template strand must be organized from 3’ to 5’ - Unwinds DNA ahead of time and rewinds the DNA that has already been transcribed - Nucleotide sequence of RNA is the same as the nontemplate strand of DNA, except T’s are replaced with U’s o Nontemplate of DNA = coding strand Chapter 8 – RNA: Transcription and Processing Stages of Transcription - Because DNA is a continuous sequence, transcriptional machinery must be directed to the start of a gene and continue down the length until the end - Three distinct stages: 1. Initiation in Prokaryotes ▪ RNA polymerase binds to DNA promotor close to start of transcribed region – also called the 5’ regulatory region ▪ Non template is usually shown because they are in the same orientation ▪ Promotor is upstream of initiation site (5’ end of gene; downstream is later in the transcription process ▪ Upstream nucleotides are indicated by (-) and downstream by (+) ▪ First DNA base to be transcribed is +1 Chapter 8 – RNA: Transcription and Processing This figure shows an E. coli gene and 7 different promotors: -35 and -10 regions have great similarities (35 and 10 base pairs upstream of first transcriptional base) - Do not have to be identical to arrive at same set of nucleotides: consensus sequence - RNA polymerase holoenzyme binds to DNA here, unwinds, and begins RNA synthesis - Intervening part is 5’ untranslated region (5’ UTR) ------------------------------------------------------------------------------------------------------------------------------- ▪ RNA polymerase holoenzyme scans the DNA for a promotor region • Two alpha subunits, one beta, one beta’ and one w • Also includes sigma factor ▪ Alpha: assemble enzyme; promote interaction with regulatory proteins ▪ Beta: active in catalysis ▪ Beta’: binds to DNA ▪ W: enzyme assembly and gene expression ▪ Sigma: binds to -10 and-35 regions; separates DNA strands around -10; dissociates from rest of complex after initiation • Other sigma factors can recognize different promotor sequences 2. Elongation in Prokaryotes ▪ RNA polymerase unwinds DNA ▪ Region of single stranded DNA is called transcription bubble where template strand is exposed and polymerase monitors addition of ribonucleoside triphosphate to DNA template if it matches • Energy needed for this is derived from splitting high-energy triphosphate ▪ Rewinds DNA that has already been transcribed Chapter 8 – RNA: Transcription and Processing 3. Termination in Prokaryotes ▪ Continues past protein-encoding segment of the gene, creating a 3’ untranslated region (3’ UTR) at the end of transcript ▪ Continues until termination signal reached ▪ Two major mechanisms for termination of E. coli • Intrinsic is direct: terminator sequences are 40bp, ending in GC rich region followed by strong of 6 or more A’s o Because this will result in CG rich area in RNA, the bases can bond with each other (internally) forming a hairpin loop – GC stems are more stable than AU o Loop followed by string of U’s o Strength of hybrid (DNA-RNA) in bubble is determined by amount of G-C pairs compared to A-U; will backtrack o Polymerase pauses after U’s, but will encounter the hairpin so, releases RNA from polymerase and polymerase from DNA • Rho dependent: this is a protein that recognizes nucleotides that act as termination signals for RNA polymerase o Do not have hairpin or string of U’s at 3’ end o 40-60 nucleotides rich in C’s, poor in G’s and have upstream rut site (rho utilization) o Bind nascent RNA chain at the rut site, upstream from sequences where RNA polymerase pauses o Rho factors facilitate release of RNA from polymerase o So: binding of rho to rut, pause, rho-mediated dissociation ^ Rho binding to rut 8.3 Transcription in Eukaryotes 1. Larger eukaryotic genome = more genes to be recognized and transcribed a. Much more non-coding DNA in eukaryotes; genes farther apart b. Makes initiation step much more complicated; for multicellular eukaryotes, finding the start of the gene can be very difficult i. Have 3 RNA polymerases for I – rRNA genes, II – protein encoding genes and III – small functional RNA’s ii. Require assembly of many proteins at a promotor before RNA poly. II can begin to synthesize RNA; some proteins called general transcription factors (GTFs) 2. Presence of a nucleus in eukaryotes a. Transcription and translation are spatially separated – transcription in the nucleus and translation in the cytoplasm b. Before it leaves the nucleus, primary transcript must be modified 3. DNA for transcription is organized into chromatin in eukaryotes (naked in prokaryotes) a. Regulates eukaryotic gene expression Chapter 8 – RNA: Transcription and Processing Transcription Initiation in Eukaryotes - Starts when sigma subunit of RNA polymerase holoenzyme recognizes -10 and -35 o Subunit will dissociate afterward, but transcription continues inside bubble
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