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Gene expression:
production of a particular mRNA from a gene (DNA sequence)
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Changes in gene expression are involved in:
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-Response of cells to their environment
-Regulation of cell cycle
- -Distinct activities of different cell types in tissue and organ function
- -Cell differentiation and development
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Variable Gene Expression:
Response of cells to their environment
example:
-Heat shock protein genes are expressed when cells exposed to elevated temperatures
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Variable Gene Expression:
Regulation of cell cycle
example:
Mutations causing a reduction in expression of tumour suppressor genes such as Rb or p53 is a major contributing cause in some cancers
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Variable Gene Expression:
Distinct activities of differentiated cell types and co-ordination of different cell types in tissue and organ function
example:
-Mutation that changes the regulation of the human apolipoprotein A-I gene promoter (encodes a serum lipid transport protein) affects blood LDL/HDL ratios
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Variable gene expression:
Cell differentiation and development
example:
Many genes are expressed only once during the life of an organism, for only a short period of time during embryonic development in only one or a few cell types, yet are critical for proper formation of the adult form
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RNA polymerases
- -principal enzymes responsible for RNA synthesis (transcription)
- –synthesize RNA from a DNA template.
- -complex enzymes made up of multiple polypeptide chains.
- -Do not require a primer sequence to initiate RNA synthesis
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promoters
- -the DNA sequence with which RNA polymerase and other proteins interact to initiate and regulate transcription of a gene
- -molecular dimmer switch
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Transcription
RNA synthesis using a DNA template
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Translation
protein synthesis using an mRNA template
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Transcription in Eukaryotes vs Prokaryotes
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Chromatin: In eukaryotes, takes place on DNA that has chromatin (nucleosomes must be displaced), takes place on free DNA in prokaryotes
Compartmentalization:
Extensive RNA Processing
Multiple nuclear RNA polymerases in euk:
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eukaryotic RNA polymerase
- an enzyme that catalyzes the synthesis of RNA from a DNA template. Three different nuclear RNA polymerases each responsible for different cellular RNAs
- RNA polymerase I
- RNA polymerase II
- RNA polymerase III
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In prokaryotes there is _ RNA polymerase
1
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RNA polymerase I
Ribosomal RNA (rRNA): 5.8S, 18S, 28S
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RNA polymerase II
ALL Messenger RNAs (mRNAs)
-smallest proportion of RNA but greatest regulatory complexity
-regulatory mechanisms have evolved to ensure that the correct gene is transcribed in the correct cell type at the correct time and in the correct amount
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RNA polymerase III
Ribosomal RNA (rRNA): 5S
ALL Transfer RNAs (tRNAs)
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rRNAs and tRNAs: make up well over __% of the total RNA (by mass) in any given cell
90
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DNA sequence element
-term used to denote a specific short sequence of DNA base pairs within a gene promoter for which a specific functional property with respect to regulation of transcription of that particular gene has been ascertained
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•Cis-acting DNA sequences
(also called promoter and enhancer elements) regulate expression of genes
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molecular dimmer switch
- -combination of DNA sequence element and cis-acting DNA sequences
- -regulate transcription
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•Basal or core promoter
Genes transcribed by RNA polymerase II contain core promoter elements, including the TATA box (others also exist), and an initiator (Inr) DNA sequence
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•Upstream promoter, and enhancers
transcriptional regulatory sequences that can be located at a significant distance from the promoter.
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reporter genes
a gene that researchers attach to a regulatory sequence of another gene of interest
- example:
- -(GFP) causes cells that express it to glow green under blue light
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Transcription factors
2 categories
-proteins that are required for RNA polymeraseII to initiate transcription.
- -General transcription factors
- -gene-specific transcription factors
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General transcription factors
Found where?
- -involved in transcription from all polymerase II promoters.
- -constitute part of the basic (or general) transcription machinery
- -involved in formation of the transcription initiation complex
-Found in the core or basal promoter
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gene-specific transcription factors
found where?
- -transcription factors that are not part of the general machinery.
- -bind to promoter and enhancer elements (with specific DNA sequences) of one or a small number of different genes,
- -direct activity of the general transcription factors
-found in upstream promotor
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How do gene specific transcription factors work
by either
-modifying the rate of transcription initiation by the basal transcription complex,
and/or
-the rate of basal transcription complex assembly
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Where does the transcription initiation complex/pre-initiation complex bind?
core or basal promoter
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TATA box
a regulatory DNA sequence found in the promoters of many eukaryotic genes transcribed by RNA polymerase II
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TATA-binding protein, or TBP,
is a basal transcription factor that binds directly to the TATA box (DNA sequence-specific binding properties).
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•TBP-associated factors, or TAFs,
- polypeptides associated with TBP in the general transcription factor, TFIID.
- (transcription factor II D)
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•TFIIB
associates with TFIID at the core promoter,
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Steps to formation of inititiation complex
- -TBP binds to the TATA box
- -TFIID associates with TBP
- -TFIIB associates with TFIID
- -Recruitment of RNA polymerase and TFIIF
- -Binding TFIIE and TFIIH
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proximal and distal promoter elements
-binding sites for gene specific transcription factors that modulate activity of the basal transcription complex
-same as enhancers
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the recognition and binding sites of most transcription factors consist of short DNA sequences, typically spanning __-__ base pairs
6–10
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Two example transcription factors
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What specifies the amount of transcriptional initiation by RNA pol II at a gene in a particular cell type?
a combination of transcription factors interacting at a specific gene’s promoter/enhancer sequences at any one point in time
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DNA-affinity chromatography
a method used to isolate transcription factors based on their binding to specific DNA sequences in order to study them further
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Steroid hormone receptors
(contain a zinc finger domain) are transcription factors that regulate gene transcription in response to hormones such as estrogen and testosterone
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•Homeodomain proteins
(contain a helix-turn-helix DNA binding domain) play critical roles in the regulation of gene expression during embryonic development.
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Reporter gene:
- -a gene that is easily visualized (once it is translated into a protein) whose gene sequence is placed upstream of the gene whose expression you actually want to visualize
- -sometimes placed using plasmids
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