Term
| Prokaryotic RNA Polymerase requires |
|
Definition
| sigma factor specific to the promoter site |
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Term
| Core Prokaryotic RNA Polymersase |
|
Definition
competent for transcriptional elongation, but not able to recognize promoter
has 2 sigma pieces and Beta and Beta prime pieces |
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Term
| Prokaryotic RNA Polymerase Holoenzyme |
|
Definition
| competent for transcription initiation; sigma factor dissociates after new RNA strand reaches ≈10 nt |
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Term
|
Definition
Sigma factor directly contacts the DNA at the promoter site in the -35 and -10 regions.
This binding only occurs when sigma factor is present in the holoenzyme complex; not when it is ‘free’ in the cell.
-alternative sigma factors associate with same core RNA polymerase, but recognize distinct promoters |
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Term
Lactose regulation
Glucose and Galactose |
|
Definition
Operator – short regulatory region within the promoter region. It is to this operator region that activator or repressor will bind and turn the gene on or off.
Note: lac repressor only binds to its operator in the absence of its inducer (lactose)
DNA footprinting experiment comparing the binding sites for RNA polymerase and the lac repressor near the transcriptional start site (+1) for the lac operon
The lac repressor binds to the lac operon at a site overlapping the promoter and blocks binding by RNA polymerase |
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Term
|
Definition
The trp repressor binds to its operator only in the presence of tryptophan (co-repressor)
Binding of tryptophan to the trp repressor causes a minor conformational change
Repositioning of two critical helical segments of the trp repressor upon binding its co-repressor allow it to bind to the DNA helix |
|
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Term
| In prokaryotes, genes encoding enzymes of a specific biochemical pathway are often clustered into an |
|
Definition
| operon that is transcribed from a single promoter site |
|
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Term
| The transcriptional product of an operon is a |
|
Definition
polycistronic mRNA (encoding multiple proteins).
Each coding sequence has its own translational initiation and termination site.
Clustering of genes into an operon provides a means of coordinately controlling expression by regulating initiation from a single promoter |
|
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Term
| Lac Operon codes 3 genes required for utilization of lactose as an energy source |
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Definition
| Beta-Galactosidase, Lactose permease, Thiogalactoside transacetylase |
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Term
| The trp operon encodes 5 genes required for the biosynthesis of |
|
Definition
| the amino acid tryptophan |
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Term
| One fundamental strategy for regulating transcription: Negative regulation |
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Definition
| In the absence of any regulatory factors, the gene is transcribed (on). Control involves a repressor that inhibits transcription only under certain conditions. |
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Term
| One fundamental strategy for regulating transcription: Positive regulation |
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Definition
| In the absence of any regulatory factors, the gene is not transcribed or transcribed weakly. Control involves an activator that increases the ability of RNA polymerase to bind to promoter. |
|
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Term
|
Definition
| increased [protein] due to increased transcription of the gene |
|
|
Term
Promoter and Operator switches are important for gene switches
Promoter –
Operator – |
|
Definition
Promoter – Specific DNA sequence to which RNA polymerase binds and initiates transcription
Operator – short regulatory region within the promoter region. It is to this operator region that activator or repressor will bind and turn the gene on or off. |
|
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Term
| Genes required for the metabolism of alternative carbon sources in E. coli are subject to catabolite repression: their transcription does not occur when glucose is available |
|
Definition
E. coli grown in the presence of lactose and glucose do not transcribe the lac operon.
Glucose is used in preference to any other carbon source (catabolite repression); only when glucose is depleted are other compounds metabolized.
Catabolite repression is controlled by the Catabolite Activator Protein (CAP) and cAMP, which binds to CAP. |
|
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Term
| Catabolite repression is controlled by |
|
Definition
the Catabolite Activator Protein (CAP) and cAMP, which binds to CAP.
CAP binds to the lac operon at an upstream site immediately adjacent to the promoter |
|
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Term
| The synthesis of cAMP by adenylyl cyclase is increased in bacteria when glucose is depleted from its environment |
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Definition
| cAMP is an intracellular signal that glucose supplies have been exhausted and the cell should use alternative C sources |
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|
Term
Lac operon combines both positive and negative controls
Transcription of the lac operon requires both the presence of lactose and absence of glucose |
|
Definition
POSITIVE CONTROL is through the CAP
Glucose decreases
cAMP increases
cAMP binds to CAP and turns transcription of lac Z “ON”.
NEGATIVE CONTROL is through the lactose repressor.
Lactose levels are low, the repressor binds to the operator and ensures that the transcription is TURNED OFF. |
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Term
|
Definition
| Operon Off because CAP not bound |
|
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Term
|
Definition
| Operon Off both because Lac repressor bound and because CAP not bound |
|
|
Term
|
Definition
| Operon Off because Lac repressor bound |
|
|
Term
|
Definition
|
|
Term
|
Definition
| bends DNA and helps to recruit RNA polymerase |
|
|
Term
| Transcriptional activators |
|
Definition
| can act at a distance to increase initiation by a DNA looping mechanism |
|
|
Term
Alternative sigma factors that share the ability to bind to core RNA polymerase, but differ in their specificity of DNA binding, can direct transcription to unique sets of genes
Genes encoding enzymes of a specific biochemical pathway are often clustered in the chromosome as an operon transcribed coordinately from a single promoter
Studies have shown that the lac operon has a repressor protein that inhibits transcription in the absence of lactose
This lac repressor binds to the operator and overlaps the promoter site to block RNA polymerase binding.
Binding of repressors is controlled by small ligands. |
|
Definition
Genes involved in metabolism of alternative carbon sources in E. coli are subject to catabolite repression; their synthesis is prevented when glucose is available.
Catabolite repression is controlled by the Catabolite Activator Protein (CAP) which binds to cAMP; cAMP levels increase when glucose is depleted.
The cAMP-CAP complex binds to a DNA site immediately upstream of the promoter to enhance the ability of RP to bind to the weak promoters in operons encoding metabolic enzymes for alternative carbon sources.
Transcriptional repressors and activators can influence promoter utilization when binding to sites distant (several 100 bp) from the promoter; DNA looping allows such factors to contact proteins binding directly to the promoter. |
|
|
Term
Promoter
Sigma Factor
DNA footprint
Consensus sequence
Promoter strength
Gene induction
Repressor
Co-repressor
Catabolite repression
Activator/Enhancer
DNA looping
Operon |
|
Definition
|
|
Term
| Regulation of gene expression in eukaryotic organisms is a complex process |
|
Definition
Eukaryotic cells generally have more genes and a lot more DNA than prokaryotic cells.
DNA in eukaryotic cells are packaged into complex chromosomal structures that limit accessibility of the transcriptional machinery.
Metazoan organisms possess multiple cell types with specialized function determined by differences in gene expression.
Development and differentiation are largely regulated by temporal and spatial changes in gene expression.
Many cell signaling pathways affect cell function and physiology by altering patterns of gene expression |
|
|
Term
| Regulation of gene expression in eukaryotic organisms is a complex process |
|
Definition
Eukaryotic cells generally have more genes and a lot more DNA than prokaryotic cells.
DNA in eukaryotic cells are packaged into complex chromosomal structures that limit accessibility of the transcriptional machinery.
Metazoan organisms possess multiple cell types with specialized function determined by differences in gene expression.
Development and differentiation are largely regulated by temporal and spatial changes in gene expression.
Many cell signaling pathways affect cell function and physiology by altering patterns of gene expression |
|
|
Term
| Eukaryotic Cells Have Multiple Forms of RNA Polymerase |
|
Definition
Prokaryotic cells contain a single RNA polymerase that is responsible for transcribing all classes of RNA.
Eukaryotic cells possess multiple forms of RNA polymerase that are specialized to transcribe different classes of RNA.
These multiple forms of RNA polymerase were first discovered by fractionating ‘nuclear extracts’ by ion exchange chromatography.
Fractionation of Nuclear Proteins by Ion-Exchange Chromatography Reveals Multiple Forms of Eukaryotic RNA Polymerase |
|
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Term
|
Definition
| messenger RNAs, code for proteins |
|
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Term
|
Definition
| ribosomal RNAs, form the basic structure of the ribosome and catalyze protein synthesis |
|
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Term
|
Definition
| transfer RNAs, central to protein synthesis as adaptors between mRNA and amino acids |
|
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Term
|
Definition
| microRNAs, regulate gene expression typically by blocking translation of selective mRNAs |
|
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Term
|
Definition
| small interfering RNAs, turn off gene expresson by directing degradation of selective mRNAs and the establishment of compact chromatic structures |
|
|
Term
| alpha-amanatin - a differential inhibitor of eukaryotic RNA polymerases |
|
Definition
alpha-amanatin - a differential inhibitor of eukaryotic RNA polymerases
inhibits RP II activity at low concentrations (~1 mg/ml)
inhibits RPIII activity at high concentrations (~30 mg/ml) |
|
|
Term
Cells grown in the presence of low concentrations of -amanatin synthesize pre-rRNA and tRNAs, but not pre-mRNA. (RPII is inhibited at low conc.)
Thus, RPII is responsible for transcribing ___________ |
|
Definition
| Pre-mRNA (protein coding genes) & some snRNAs |
|
|
Term
Cells grown in the presence of high concentrations of -amanitin synthesize only pre-rRNA. (RPIII is inhibited at high conc.)
Hence, RPIII is responsible for transcribing ___________ |
|
Definition
| tRNA, 5S rRNA, some snRNAs & other small RNAs |
|
|
Term
| RPI is responsible for transcribing ____________ |
|
Definition
| Pre-rRNA (5.8S, 18S & 28S rRNAs) |
|
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Term
| The Conserved TATA Box Found Upstream from Many RP II Start Sites |
|
Definition
| The TATA box is located at -35 to -25 in eukaryotic promoters. |
|
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Term
| Eukaryotic RNA polymerases require additional components for recognition of their promoter and initiation |
|
Definition
Like the core enzyme of prokaryotes, purified RNA polymerases can catalyze RNA elongation, but are unable to initiate transcription from their proper promoter sites.
However, a ‘nuclear extract’ containing RNA polymerase and other nuclear proteins can do this.
Additional proteins in the nuclear extract that are needed are called ‘general transcription factors’.
Five general transcription factors are required for promoter recognition and initiation by RP II. |
|
|
Term
| General transcription factors (GTFs) are named based on the form of RNA polymerase that they work with and an arbitrary letter |
|
Definition
TFIIX
Transcription Factor for:
RNA polymerase II
A, B, C, etc. based on order of discovery |
|
|
Term
|
Definition
Recognizes TATA box; TFIIB recruitment
TFIID is the first factor to bind to the promoter and nucleates formation of the initiation complex
TFIID is composed of a subunit designated the TATA Binding Protein (TBP) and 11 additional subunits call TAFs (TBP-associated factors)
TBP binds to the TATA box by itself recognizing the minor groove of DNA helix and causing a bend in the DNA |
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|
Term
|
Definition
| Recognizes TFIID at promoter; positions RPII at promoter site |
|
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Term
|
Definition
| 12 subunits - Catalytic Machinery |
|
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Term
|
Definition
| Helicase activity to unwind the DNA at the transcription site; another subunit has protein kinase activity that phosphorylates CTD of RPII and releases RNA polymerase from the promoter |
|
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Term
| Structure of Complex between TBP and a TATA Box-containing Promoter Fragment |
|
Definition
Note: DNA is distorted by ~80° at TATA consensus upon binding TBP
This distortion brings DNA sequences on both sides of the distortion together to allow for subsequent protein assembly steps |
|
|
Term
| Initiation of transcription by RNA polymerase II requires a set of general initiation factors |
|
Definition
The subunit TBP of TFIID recognizes and binds the TATA box, which then enables the adjacent binding of TFIIB.
The rest of the transcription factors, as well as RNA polymerase II itself, assemble at the promoter.
TFIIH then uses ATP to pry apart the DNA double helix at the transcription start site.
TFIIH also phosphorylates CTD of RNA pol II changing its conformation so that the polymerase is released from the general factors and begins transcription elongation.
CTD – C-terminal domain |
|
|
Term
| The Carboxy Terminal Domain (CTD) of RNA Polymerase II |
|
Definition
The largest subunit of RPII contains at its C-terminal domain a repeating heptapeptide:
(YSPTSPS)n where n=27-52
The CTD is found exclusively on RPII; not RPI or RPIII or any prokaryotic RP
The CTD is essential for cell viability
The CTD is subject to phosphorylation at several of its serine residues |
|
|
Term
| Polymerase II requires activator, mediator and chromatin modifying proteins for its activity |
|
Definition
Transcriptional activators or Enhancers – These have activation domain and binding domain
Mediator
Chromatin modifying enzymes – These allow greater access to the DNA present in chromatin. |
|
|
Term
| The Mediator is a very large protein complex that works together with GTF’s and RPII to form the preinitiation complex |
|
Definition
| Mediator is a 26 subunit complex that coordinates preinitiation complex formation and activation of transcription by activators |
|
|
Term
| Some surprising properties of transcriptional enhancers |
|
Definition
Enhancers can function at great distances (up to 50 kb) from the basal promoter.
Enhancers are found either upstream or downstream of the basal promoter that they influence in different genes.
Enhancers can (usually) function when artificially linked to a promoter from a different gene.
Enhancers function at great distances because the DNA between the enhancer and the promoter site can loop |
|
|
Term
| Control of gene expression in eukaryotes is critical for |
|
Definition
| cell specialization and the ability of a cell to respond to external and internal environmental changes. |
|
|
Term
| DNA microarrays allow the |
|
Definition
| rapid comparison of patterns of gene expression between two cells or a given cell under different conditions. |
|
|
Term
| Eukaryotic cells have three forms of RNA polymerase specialized to transcribe |
|
Definition
| pre-rRNA (RP I), pre-mRNA (RP II) and small stable RNAs (RP III). |
|
|
Term
| Promoters for RP II contain |
|
Definition
| short conserved DNA elements found in some, but not all, promoters - the TATA box is the most common of these elements. |
|
|
Term
| All eukaryotic RNA polymerases require |
|
Definition
| additional components (General Transcription Factors) for recognition of their promoters and initiation. |
|
|
Term
| Five GTFs are required for initiation by |
|
Definition
| RP II and these factors function in vitro in an ordered, stepwise fashion to form a functional initiation complex. |
|
|
Term
| TFIID is the first transcription factor to bind to the basal promoter |
|
Definition
| different subunits of TFIID recognize distinct core promoter elements. |
|
|
Term
| TFIIH is the last of the GTFs to bind to the initiation complex and contains both |
|
Definition
| protein kinase and DNA helicase activities. |
|
|
Term
| The helicase activities of TFIIH help |
|
Definition
| to melt the DNA helix at the transcriptional start site, allowing RNA polymerase II access to DNA base pairing information. |
|
|
Term
| The C-terminal domain (CTD) of RNA polymerase II consists of |
|
Definition
| a conserved repeating heptapeptide that is either unphosphorylated ( in initiating RP II) or phosphorylated (in elongating RP II). |
|
|
Term
| The protein kinase subunit of TFIIH phosphorylates |
|
Definition
| the CTD and allows release of RPII from the initiation complex and recruitment of pre-mRNA modification complexes. |
|
|
Term
| The Mediator is a large protein complex with 26 subunits that helps to |
|
Definition
| coordinate preinitiation complex formation and interfaces between activators and the basal txn machinery. |
|
|
Term
| Enhancers can act at great distances and be found either upstream or downstream from the transcription start site |
|
Definition
| transcription factors binding at enhancer can contact the basal transcription machinery through DNA looping. |
|
|
Term
| DNA in a eukaryotic cell is packaged into nucleosomes and higher order structures that impede access of TFIID & RNA polymerase II to the DNA. |
|
Definition
Cells possess a number of protein/enzyme complexes that can move and remove nucleosomes, mark chromatin, or propagate a mark along the chromatin.
These chromatin remodeling and modifying complexes work in conjunction with activators and repressors to control gene expression. |
|
|
Term
| DNA in eukaryotic cells is packaged into complex chromosomal structures that can serve as impediments to binding of |
|
Definition
| RNA polymerase II and the GTFs. |
|
|
Term
| Major and minor grooves of the DNA helix are accessible along the surface of the nucleosome |
|
Definition
- The major groove of the DNA is accessible to DNA binding proteins.
- The N-terminal histone tails can be marked by chemical modifications. |
|
|
Term
| Transcriptional activators can bind to DNA sites within nucleosomes to |
|
Definition
| initiate chromosomal changes necessary for transcriptional initiation |
|
|
Term
| How do activators deal with nucleosomes |
|
Definition
Once bound to their binding sites, some activators can rearrange nucleosomes to provide access to other proteins.
More commonly, activators act by recruiting coactivators - chromatin remodeling complexes and chromatin modifying complexes - that alter the nucleosomal structure or organization. |
|
|
Term
| How do activators deal with nucleosomes |
|
Definition
Once bound to their binding sites, some activators can rearrange nucleosomes to provide access to other proteins.
More commonly, activators act by recruiting coactivators - chromatin remodeling complexes and chromatin modifying complexes - that alter the nucleosomal structure or organization. |
|
|
Term
| Mechanisms used by transcriptional activators to direct local alterations in chromatin structure |
|
Definition
The first three mechanisms show that chromatin remodeling complexes move, remove or exchange octamer cores. All this makes DNA more accessible to RNA polymerase and GTFs
The fourth mechanism shows that patterns of histone modifications can be altered.
remodel the nucleosomes
remove the histones
replace the histones
specific pattern of histone modification |
|
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Term
| Chromatin remodeling complexes phase or remodel nucleosomes |
|
Definition
Chromatin remodeling complexes were first identified as transcriptional coactivators that are recruited by different activators.
Chromatin remodeling complexes contain many subunits and use energy of ATP hydrolysis to displace or remodel nucleosomes.
Chromatin remodeling complexes also contain DNA helicase activity.
Once acted upon, nucleosomes remain in a ‘remodeled’ state for some time after dissociation of the chromatin remodeling complex. |
|
|
Term
| Chromatin Remodeling Complexes |
|
Definition
| Cells possess a number of chromatin remodeling complexes. All contain multiple subunits. All hydrolyze ATP to slide or displace nucleosomes. |
|
|
Term
| Chromatin Remodeling Complexes |
|
Definition
| Cells possess a number of chromatin remodeling complexes. All contain multiple subunits. All hydrolyze ATP to slide or displace nucleosomes. |
|
|
Term
| Amino-terminal histone tails |
|
Definition
| extend out from core nucleosome particle |
|
|
Term
| Covalent modifications found on histones –The histone code hypothesis |
|
Definition
| The particular postranslational modifications found in different regions of the chromatin constitute a “histone code”. This influences the chromatin function by creating or removing binding sites for chromatin-associated proteins. |
|
|
Term
| Covalent modifications found on histones –The histone code hypothesis |
|
Definition
| The particular postranslational modifications found in different regions of the chromatin constitute a “histone code”. This influences the chromatin function by creating or removing binding sites for chromatin-associated proteins. |
|
|
Term
| Histone acetylation and transcription |
|
Definition
Acetylation of amino-terminal ‘tails’ of four core histones is highly dynamic in the cell.
Nucleosomes associated with actively transcribed genes contain higher levels of acetylated histones than nucleosomes associated with inactive genes.
Acetylation neutralizes a positive charge on lysine and this may reduce interaction between linker DNA and adjacent nucleosome particle.
Acetylation of the epsilon-amino of lysine residues in the N-terminal histone tails |
|
|
Term
| Many transcriptional coactivators possess HAT activity |
|
Definition
When first HAT was purified (1996) and its cDNA cloned, it turned out to be homologous to a subunit of a yeast coactivator known as GCN5.
Subsequently, many other transcriptional coactivators were found to possess HAT activity.
Hence, by recruiting coactivators to specific promoter regions, activators can cause a change in acetylation of histone tails and alter chromatin accessibility for other transcriptional factors. |
|
|
Term
| Coactivators can alter acetylation of nucleosomal histones |
|
Definition
GCN4 is the activator
GCN5 is the coactivator |
|
|
Term
| Multiple HAT-containing Coactivator Complexes Occur in All Cells |
|
Definition
Twenty different HAT-containing coactivators complexes have been identified in yeast.
All HAT-containing coactivator complexes consist of multiple subunits.
The same HAT catalytic subunit can be part of several coactivator complexes.
HAT-containing coactivator complexes are conserved from yeast to humans. |
|
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Term
| Chromatin Remodeling Complexes |
|
Definition
Function as transcriptional coactivators
Multisubunit complexes
Utilize ATP hydrolysis to alter histone-DNA contacts
Can phase nucleosomes or displace histones |
|
|
Term
| Chromatin Remodeling Complexes |
|
Definition
Function as transcriptional coactivators
Multisubunit complexes
Utilize ATP hydrolysis to alter histone-DNA contacts
Can phase nucleosomes or displace histones |
|
|
Term
| Chromatin Modifying Complexes |
|
Definition
Function as transcriptional coactivators
Multisubunit complexes
Covalently modify histone tails by acetylation, methylation, etc
Establish ‘histone code’ |
|
|
Term
| Transcriptional activation is a multistep process that involves chromatin remodeling complexes and coactivators functioning sequentially |
|
Definition
This example shows the order with which activators, chromatin remodeling and chromatin modifying complexes function on one particular gene. This order can vary from gene to gene
-Gene activator protein binds to chromatin
-Chromatin remodeling
-Covalent histone modification
-Additional activator proteins bound to gene regulatory region
-Assembly of pre-initiation complex at the promoter
-Transcription intiation |
|
|
Term
| Many corepressor complexes contain histone deacetylase activity |
|
Definition
| Deactylation of the histone tails in the vicinity of UME6 inhibits binding of GTFs and thus inhibits transcription |
|
|
Term
| Gene inactivation by histone deacetylation |
|
Definition
Transcription in initiated by interactions between activators, coactivators, and RNA Polymerase
Repressors displace the activators, and a corepressor inhibits RNA polymerase
A histone deacetylase associates with the corepressor, and RNA Polymerase dissociates from DNA
Histone deacetylaation leads to chromatin formation, further repressing transcription of the gene |
|
|
Term
| HDACs are associated with longevity |
|
Definition
The most famous HDAC is the yeast SIR2 HDAC and its mammalian ortholog SIRT1 (collectively called sirtuins).
Deletion of SIR2 in yeast shorten their lifespan and addition of extra copies extend their lifespan.
Caloric restriction in all organisms increases longevity and depends on sirtuins (Results from a research done 10 years ago).
A component of red wine, resveratrol, activates sirtuins. |
|
|
Term
|
Definition
| The multiple and specific modifications of the histone tails provide a code that imparts information not just for transcription but for DNA repair and chromatin condensation as well |
|
|
Term
|
Definition
| proteins that have domains that bind the modified residues |
|
|
Term
| The protein domain that binds acetylated lysine is called a |
|
Definition
bromodomain.
Stabilized open state |
|
|
Term
| The protein domain that binds methylated lysine is called a |
|
Definition
chromodomain.
Stabilized closed state |
|
|
Term
| Code readers may themselves be transcription factors or may bind other factors that affect transcription |
|
Definition
| code reader complex attachment to other components in nucleus may lead to gene expression, gene silencing, or other biological function |
|
|
Term
| Code readers may be coupled with writer complexes that propagate the signal |
|
Definition
| The writer collaborates with the a code-reader protein to spread its mark from nucleosome to nucleosome by means of this reader-writer complex. |
|
|
Term
| In addition to site-specific actions of activators and repressors, longer-range effects on chromatin structure are critical for determining gene accessibility for transcription |
|
Definition
Microscopically, chromatin can be divided into:
Euchromatin: more lightly staining; in a more open, accessible conformation
Heterochromatin: more densely staining; in a more compacted, inaccessible conformation
Actively transcribed genes are found in euchromatin.
Centromeres, telomeres and highly repetitive DNA are found in heterochromatin.
Some regions of chromatin are euchromatic in one cell type (expressed), but heterochromatic on others (repressed), contributing to differences in transcriptional activity. |
|
|
Term
|
Definition
| activity of a gene depends on its position relative to a nearby heterochromatin on a chromosome |
|
|
Term
| Activators can bind to their recognition sites in nucleosome-associated DNA |
|
Definition
| and displace or modify nucleosomes to allow access for other gene regulatory proteins. |
|
|
Term
| Activators can bind to their recognition sites in nucleosome-associated DNA |
|
Definition
| and displace or modify nucleosomes to allow access for other gene regulatory proteins. |
|
|
Term
| Transcriptional coactivators are often |
|
Definition
| chromatin modifying or histone modification complexes that act to alter chromatin structure or organization. |
|
|
Term
| Chromatin remodeling complexes utilize the energy of |
|
Definition
| ATP hydrolysis to displace or phase nucleosomes along a stretch of DNA. |
|
|
Term
| Chromatin modifying complexes possess |
|
Definition
| histone acetyl transferase activity and act by acetylating nucleosomal histones to weaken histone-DNA interactions. |
|
|
Term
| Different chromatin modifying and remodeling complexes act |
|
Definition
| sequentially at a specific gene to activate transcription. |
|
|
Term
| Corepressors function in part by |
|
Definition
| deacetylating nucleosomal histones to increase nucleosome stability and reduce access of transcriptional machinery. |
|
|
Term
| Methylation of lysine residues is another histone modification often associated with |
|
Definition
|
|
Term
| The pattern of histone modifications is critical for |
|
Definition
| determining the function of the packaged DNA and is known as the histone code. |
|
|
Term
| The histone code is read by |
|
Definition
| proteins (code readers) that possess domains recognizing specific amino acid modifications. |
|
|
Term
| Specific histone codes may be propagated along the DNA by |
|
Definition
| code writer proteins to establish chromatin regions that are repressed or active. |
|
|
Term
| Active (euchromatic) and inactive (heterochromatic) regions of chromatin are established by |
|
Definition
|
|
Term
|
Definition
| small nuclear RNAs, function in a variety of nuclear processes, including the splicing of pre-mRNA |
|
|
Term
| rRNAs are named by their S-value, or sedimentation in an ultracentrifuge |
|
Definition
RNA Polymerase I transcribes rRNA genes:
5.8S
18S
28S |
|
|
Term
|
Definition
| all protein-coding genes, plus snoRNA genes, miRNA genes, siRNA genes, and most snRNA genes |
|
|
Term
|
Definition
| tRNA genes, 5S rRNA genes, some snRNA genes, and genes for other small RNAs |
|
|
Term
| RNA polymerase II transcribes genes that will be translated into proteins |
|
Definition
RNA pol II is composed of 12 subunits.
The polymerizing subunit has a C-terminal domain (CTD) that binds to factors that help the enzyme transcribe and process mRNAs. C-terminal tail becomes phosphorylated and changes conformation, cannot stay bound to TATA box and starts elognation – only in RNA Polymerase II
The CTD of RNA pol II has a seven amino acid repeating primary sequence (YSPTSPS)n where n=27-52). Phosphorylates serine |
|
|
Term
Messenger RNA processing in bacteria
***Bacteria have only 1 RNA polymerase
But, Eukaryotic cells: |
|
Definition
Messenger RNA processing in eukaryotes
3 RNA Polymerases (only II is involved in transcription
mRNA must undergo processing and then exported out of nucleus
Three things in processing:
1) 5’ cap
2) Introns spliced out
3) 3’ Poly-A tail
Nuclear export (not processing) |
|
|
Term
| Nascent transcripts are capped with a G residue (Cap) at their 5’-end |
|
Definition
The G nucleotide is attached by an unusual 5’-5’ linkage through a triphosphate bridge. (7-methylguanosine)
The G is often methylated and so are the 2’ and 3’ hydroxyls of the ribose.
Capping protects mRNAs from exonucleolytic degradation.
Before processing, called “nascent mRNA” or “mRNA transcript,” then processed, then called mature mRNA |
|
|
Term
| The capping enzyme complex tailors the 5’-end of the nascent mRNA |
|
Definition
Note that the nascent mRNA has a triphosphate at its 5’-end.
One phosphate is removed.
GTP is added with the release of PPi (pyrophosphate).
Of the triphosphate, two come from the nascent mRNA and one from the GTP.
Methyl groups are added..
Capping enzyme is Guanylyltransferase***
The capping enzymes bind to the CTD of RNA pol II. Once it is capped, the transferase disassociates and the cap-binding complex attaches to the 5'cap |
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Term
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Definition
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Term
| Sequences at the 3’-end of nascent mRNAs direct the addition of polyA |
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Definition
Nascent mRNAs are longer than the string of codons.
Sequences direct the cleavage of the RNA and addition of A residues.
The polyA protects the message and aids in translation.
For cleavage - AAUAAA is bound by CPSF.
GU rich region is bound by CstF.
CA rich region by a cleavage protein.
The polyA polymerase complex binds to the CTD of RNA pol II. Two proteins bind to the 2,5 phosphorylated CTD and recognize the termination sequences as they pass by. |
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Term
| Two proteins bind to the 2,5 phosphorylated CTD and recognize the termination sequences as they pass by. |
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Definition
CPSF - AAUAAA is bound by CPSF,
CstF - GU rich region is bound by CstF
CA rich region by a cleavage protein |
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Term
| The poly-A polymerase complex binds to the CTD of RNA pol II |
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Definition
The Poly-A polymerase is a strange RNA polymerase in that it does not require a template (but does require a primer).
There is no set length of A residues, but tails average about 250 As
in our mRNAs. |
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Term
| A protein called the poly-A binding protein covers the tail. |
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Definition
The Poly-A binding protein polymerizes along the tail.
This not only protects the 3’-end but serves as a signal during translation and mRNA transport from the nucleus. |
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Term
| Addition of the 3’ poly(A) tail to the transcript |
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Definition
RPII transcribes the poly(A) addition site
Polyadenylation factors bind the poly(A) signal, initiating mRNA cleavage
PAP synthesizes the poly(A) tail; PABP protects the tail from degradation and transport out of the nucleus |
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Term
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Definition
Most of our genes contain introns — intervening sequences.
Exons contain the coding (codon) sequences.
Introns are sequences between exons, not between genes |
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Term
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Definition
- can consume more sequence than exons
- are removed from the pre-mRNA
Exons are usually represented by boxes and introns by lines.
Many times the introns are shown as carats to indicate their eventual removal. |
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Term
| Splicing requires the participation of a set of noncoding RNA molecules to: |
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Definition
1) recognize splice signals
2) catalyze phosphodiester transfer |
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Term
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Definition
lariats
1) A specific adenine nucleotide in the intron sequence attacks the 5’ splice site and cuts the sugar-phosphate backbone of the RNA at this point.
2) The 5’ cut end of the intron becomes covalently linked to the adenine nucleotide, creating a loop in the RNA molecule.
3) The released free 3’OH of the exon sequence then reacts with the start of the next exon sequence joining the exons together and releasing the intron sequence in the shape of a lariat. |
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Term
| Signals indicate where to splice |
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Definition
GU signal on 5’ end and the AG signal on the 3’ end and the Adenine in the middle that creates the loop
AG at end of exon 1 marries up to G of exon 2
From 5’ to 3’ along the pre-mRNA:
*5’-splice donor is GU
Branch site is A
*3’-splice acceptor is AG |
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Term
| Splicing is carried out by |
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Definition
snRNPs
**snRNP: small nuclear ribonucleoprotein particle
- called snurps in the vernacular
composed of snRNAs and proteins
snRNAs are U1, U2, U4, U5 and U6 (1-6 without 3)
snRNPs are named for the snRNAs: U1, U2, U4-U6 and U5
some proteins are specific to each snRNP; others are components of all or a group of snRNPs |
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Term
Splicing is a cycle
RNA – RNA interactions |
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Definition
snRNPs enter and exit in a specific order.
U1 followed by U2 followed by the U4-U6/U5 triple snurp
Most interactions are RNA-RNA.
ATP is hydrolyzed |
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Term
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Definition
1) U1 snRNP on the 5' splice junction
2) BBP (Branch-point binding protein), U2AF (U2 Auxilliary factor) recognize the branch-point site
3) U2 snRNP displaces BBP, U2AF and forms base pairs with the branch-point site consensus sequence
4) U4/U6 and then U5 enter the reaction.
5) U2/U6 and U5 are catalytic core of spliceosome. RNA - RNA interactions: U6 and U2 base pair. U5 holds the 5’ and 3’ splice junctions together.
U2, U5 and U6 make a catalytic core. |
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Term
| Splicing probably takes place by RNA catalysis |
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Definition
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Term
| How are introns recognized? —the “not exon” hypothesis |
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Definition
Exons are, on average, between 100-200 nucleotides in length.
Introns are usually much longer
Length as a defining characteristic of introns
…even in this simplified system splicing decisions rest on complex interplays of yet to be determined parameters.
Splicing of designer exons reveals unexpected complexity in pre-mRNA splicing. |
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Term
| Is RNA splicing a wasteful event? |
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Definition
| No. RNA splicing enables the eukaryotes to increase the coding potential of their genome. |
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Term
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Definition
Approximately 75% of our genes are alternatively spliced
Tropomyosin is a protein that regulates contraction in muscle cells. The primary transcript can be spliced in different ways to produce distinct mRNAs which give rise to variant proteins.
The troponin T gene undergoes alternative splicing to include mutually exclusive exons. This means that adults and juveniles have different primary amino acid sequences of the protein called troponin T.
Calcitonin and calcitonin-related gene transcript (CGRP) have completely different protein sequences but are encoded by the same gene. |
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Term
| What are the Alternative Splicing classifications |
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Definition
1) Optional exon - skip over an exon
2) Optional Intron - might leave in a intron
3) Mutually Exclusive exons - choose one and skip another exon
4)Internal Splice sites - code part of one exon or intron |
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Term
| To protect mRNAs from exonuclease degradation, nascent mRNAs are capped by adding |
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Definition
| a G with an unusual 5’-5’ triphosphate attaching it to the 5’-end of the mRNA. |
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Term
| The capping enzymes are bound to the |
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Definition
| C-terminal domain (CTD) of RNA PolII. The CTD has a heptad repeat of amino acids where residues 2 and 5 are serines. |
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Term
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Definition
| 3’-tail of mRNAs by the PolyA polymerase that requires a primer but no template. |
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Term
| A sequence AAUAAA followed some 40-50 nucleotides later by a GU rich sequence signals |
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Definition
| ribo-endonucleolytic cleavage of the nascent mRNA. This provides the primer for the polyA polymerase. |
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Term
| A sequence AAUAAA followed some 40-50 nucleotides later by a GU rich sequence signals |
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Definition
| ribo-endonucleolytic cleavage of the nascent mRNA. This provides the primer for the polyA polymerase. |
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Term
| Components of the polyA tailing system travel with |
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Definition
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Term
| The polyA tail is bound by the polyA binding protein that protects it from |
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Definition
| degradation and coordinates ribosome recycling. |
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Term
| Genes may contain more intron nucleotides than |
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Definition
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Term
| Genes may contain more intron nucleotides than |
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Definition
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Term
| Genes may contain more intron nucleotides than |
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Definition
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Term
| Splicing signals occur at the |
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Definition
| 5’-splice donor, at the 3’-splice acceptor and at an internal branch-site in the intron. |
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Term
| While the signals are complex, the highest conservation of these sites are |
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Definition
| 5’-GU, AG-3’ and the branch site A, respectively. |
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Term
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Definition
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Term
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Definition
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Term
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Definition
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Term
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Definition
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Term
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Definition
| 5 snurps (snRNPs): U1, U2, U4-U6 and U5, each composed of specific snRNAs and proteins. |
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Term
| Splicing is a cycle which begins with the formation of the |
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Definition
| prespliceosome. The U1 snRNP recognizes the 5’-splice junction, and the U2-snRNA recognizes the branch site. |
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Term
| The U4-U6-U5 triple snRNP enters to |
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Definition
| catalyze lariat formation. |
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Term
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Definition
| guides splicing. U1 snRNA complements the 5’-splice site, U2 snRNA complements the branch site and the U6 snRNA displaces U1 snRNA in base-pairing with the 5’-splice junction. Energy in the form of ATP hydrolysis is needed to facilitate RNA-RNA interactions. |
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Term
| The catalytic core of the spliceosome is likely composed of |
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Definition
| U5 snRNA with help from the U6 and U2 snRNAs. It is believed that splicing is RNA catalyzed |
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Term
| Intron sequences may be distinguished from exon sequences by |
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Definition
| proteins that recognize only exons. We possess many pre-mRNAs that are alternatively spliced such that an exon that appears in a juvenile form of the mRNA is replaced by another exon in the adult mRNA. |
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Term
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Definition
1) mRNA and the first aminoacyl-tRNA bind the small subunit (Pre-intiation)
2) The large subunit associates to form the active ribosome (Initiation)
3) The second aminoacyl-tRNA binds (Elongation)
4) The aminoacyl group is transferred from the first to the second tRNA, and the first tRNA dissociates. Successive cycles of aminoacyl-tRNA binding and aminoacyl group transfer occur until the ribosome reaches a stop codon (Termination)
5) Ribosome recycling |
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Term
| Crick’s adaptor hypothesis |
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Definition
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Term
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Definition
Adapters
- carry amino acids as esters at their 3’-ends
contain a sequence of bases called the anticodon that can base pair with the codon of the mRNA
a class of noncoding RNAs made by RNA Pol III
~75 nucletides long
all end with CCA-3’
fold into cloverleaf by intramolecular base pairing
three loops, one containing anticodon
many modified bases (over 50 different types of tRNA modifications are known)
The cloverleaf folds into a 3-dimensional conformation of an inverted L, with the CCA-end (at 3’) holding the amino acid and the anticodon loop far apart. |
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Term
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Definition
D = dihydrouracil
Psi = pseudouracil
Y = isopentyl |
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Term
| codon - anticodon base pairing |
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Definition
| antiparallel; G:::C and A::U |
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Term
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Definition
Ester bond between the acid of the amino acid and the hydroxyl of the ribose at the 3’- end of the tRNA.
Adding a particular amino acid depends on the codon the tRNA recognizes.
ATP hydrolysis is needed to esterify tRNAs. Catalyzed by aminoacyl t-RNA synthetase.
This ester bond is a high-energy bond. No further ATP equivalents are needed to form the peptide bond. |
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Term
| The peptide grows N-C terminus |
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Definition
| The peptide bond is formed by the nucleophylic attack of the amino group of the incoming aminoacyl tRNA on the ester bond of the peptidyl tRNA. |
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Term
| amino acyl tRNA synthetase |
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Definition
acylate (esterify) amino acids to tRNAs.
- ~ 20 species
- While these enzymes must recognize the cognate tRNA, they do rely solely on the anticodon bases. |
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Term
| amino acyl tRNA synthetase |
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Definition
acylate (esterify) amino acids to tRNAs.
- ~ 20 species
- While these enzymes must recognize the cognate tRNA, they do rely solely on the anticodon bases. |
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Term
| Amino acyl tRNA synthetases proofread. |
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Definition
| These enzymes have two active sites. After the amino acid is esterified, it is switched to a second active site for proofreading. Proofreading means hydrolysis. |
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Term
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Definition
| Some anticodons can recognize more than one codon. The code is degenerate in the third (3’) base. A G in the first (5’) base of the anticodon can recognize both C and U. An I (deaminated A) can recognize both U and C. |
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Term
| Ribosomes are RNA-protein particles. |
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Definition
Prokaryotic: large 23S and 5S RNAs; small 16S RNA
50S + 30S = 70S
Eukaryotic: large 28S and 5.8S and 5S RNAs; small 18S RNA
60S + 40S = 80S |
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Term
| Ribosomes have three tRNA binding pockets |
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Definition
A = aminoacyl
P = peptidyl
E = ejection
A tRNA enters the A site, its amino acid is covalently attached to the peptide held by the tRNA in the P site, it is then moved into the P site so the growing peptide can add to the incoming amino acyl tRNA, before it is moved into the E site and ejected. |
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Term
| ????? delivers amino acyl tRNAs to the A site. |
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Definition
EF-Tu
EF - Elongation Factor
EF-Tu hydrolyzes GTP when delivery is completed |
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Term
| ????? delivers amino acyl tRNAs to the A site. |
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Definition
EF-Tu
EF - Elongation Factor
EF-Tu hydrolyzes GTP when delivery is completed |
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Term
| ???? moves the peptidyl tRNA from A to P site |
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Definition
EF-G
EF-G hydrolyzes GTP when transfer is complete |
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Term
| Fidelity during translation |
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Definition
How can three (or two if wobble occurs) base pairs be enough to insure the right tRNA anticodon pairs with the codon of the mRNA?
Kinetic proofreading - delay after delivery allows time for dissociation of the wrong tRNA
Induced fit - small rRNA changes conformation when proper anticodon - codon base pair forms |
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Term
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Definition
| There is a pause before the amino acid attached to the delivered tRNA is bonded to the growing peptide. This pause gives time for a tRNA with the wrong anticodon to dissociate. |
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Term
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Definition
| The small rRNA “holds” the tRNA anticodon to the mRNA codon. This occurs by a conformational change in the position of the small rRNA when the anticodon “seats” into the A site (correct anticodon-codon pair is formed). |
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Term
| The 23s/28s rRNA participates in peptide bond formation. |
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Definition
| The large rRNA has a specific three dimensional conformation |
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Term
| Ribosomal proteins are not direct participants in catalysis |
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Definition
Ribosomal proteins bind to the outside of the rRNA mass with protrusions into the rRNA mass.
Ribosomal proteins bind to the outside of the rRNA mass with protrusions into the rRNA mass.
No amino acid is within 1.8 nm of the site where the peptide bond is made.
RNA molecules that possess catalytic activity are known as Ribozymes. |
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Term
| RNA molecules that possess catalytic activity are known as |
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Definition
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Term
| Release factors recognize stop codons in the A site |
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Definition
No anticodon, i.e., no tRNA, complements the stop codons.
The release factor causes hydrolysis of the peptide from the tRNA in the P site in turn causing the dissociation of the ribosomal subunits |
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Term
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Definition
To initiate translation:
- the small ribosomal subunit binds to the mRNA
- a special tRNAmet is brought in (with met covalently bound) to the P site
- the initiating start codon is recognized
- the large ribosomal subunit joins the small subunit
- the tRNA with the 2nd amino acid is brought in to the A site |
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Term
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Definition
To initiate translation:
- the small ribosomal subunit binds to the mRNA
- a special tRNAmet is brought in (with met covalently bound) to the P site
- the initiating start codon is recognized
- the large ribosomal subunit joins the small subunit
- the tRNA with the 2nd amino acid is brought in to the A site |
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Term
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Definition
All reading frames begin with the start codon AUG which is also the only met codon.
AUG is the only codon for which there are two tRNAs, an initiator tRNAimet and a met tRNAmet for internal met codons.
In prokaryotes, tRNAimet holds fmet, formylated met. Thus, all prokaryotic proteins start with a blocked N-terminus. |
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Term
| start codon recognition, prokaryotes |
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Definition
In bacteria the 3’-end of the small ribosome (16 s RNA) base pairs with a sequence in the 5’-leader of the mRNA known as the Shine-Delgarno sequence. The nearest AUG is the start codon. Upstream sequence is required
Conserved 5’-AGGAGGU-3’Shine-Delgarno |
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Term
| start codon recognition, Eukaryotes |
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Definition
Kozak sequence
Optimal sequence: ACCAUGG (The bolded letters are highly preferred. The two nucleotides before the start codon are preferred to be pyrimidines, shown here as two C nucleotides
In eukaryotes, the small ribosomal subunit with met-tRNAimet starts at the 5’ cap and scans the mRNA for a start codon located within an optimal setting. |
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Term
initiation by initiation factors
IF – Initiation Factor |
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Definition
- eIF2 delivers metRNAimet to the P site.
- Together they bind to eIF4E&G that are cap binding proteins.
- The little “e” stands for eukaryotic. Similar initiation factors exist in prokaryotes.
- ATP is consumed in the search.
- eIF2 hydrolyzes GTP when contact is made with the AUG start codon.
- The large ribosomal subunit is brought in to join the small subunit. |
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Term
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Definition
- mRNAs can be translated simultaneously by more than one ribosome.
- Ribosomes cover ~ 40 nucleotides.
- An mRNA covered with multiple ribosomes is called a polysome. |
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Term
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Definition
- The ribosomal subunits dissociate when the protein is released.
- The cap binding proteins interact with the polyA binding protein.
- The physical proximity of the mRNA ends facilitates ribosome recycling. |
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Term
| Many antibiotics are translation inhibitors. |
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Definition
| Useful antibiotics often take advantage of the difference between prokaryotic and eukaryotic ribosomes |
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Term
| Translation is the process of |
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Definition
| translating the mRNA into the primary sequence of amino acids in a protein. |
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Term
| Translation requires a number of cellular components |
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Definition
| First, it requires a set of short noncoding RNAs called tRNAs. Amino acids are covalently bonded to tRNAs. The tRNAs have a cloverleaf structure. One loop has the anticodon that base pairs with the codon in the mRNA. All tRNAs end in CCA where the amino acid is esterified to the terminal 3’ OH of the A. |
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Term
| The code table shows that many amino acids have more than one codon |
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Definition
| (degeneracy). It also shows that there are three stop codons, UAA, UAG and UGA. |
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Term
| Wobble is the ability of a tRNA to |
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Definition
| recognize degenerate codons. In eukaryotes, a G or I in the anticodon is capable of recognizing both C and U. |
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Term
| Amino-acyl tRNA synthetases recognize |
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Definition
| tRNAs and esterify the cognate amino acid to the tRNA. This requires ATP hydrolysis (ATP -> AMP + PPi). |
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Term
| Ribosomes are composed of |
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Definition
| rRNA and ribosomal proteins. They mediate the base pairing between the anticodon of the charged tRNAs and the mRNA. Ribosomes are composed of a large and a small subunit. |
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Term
| Ribosomes have three tRNA binding sites |
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Definition
| the amino-acyl site (A), the peptidyl site (P) and the ejection site (E). tRNAs move from A to P to E. |
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Term
| Translation initiation begins at a |
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Definition
| methionine codon. A special tRNAmet is used. |
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Term
| All RNAs have a leader sequence before the start codon. Ribosomes find the proper start codon either by |
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Definition
| base pairing between mRNA and rRNA (prokaryotes) or by scanning from the capped 5’-end (eukaryotes) |
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Term
| Initiation factors, proteins not bound to the ribosome, |
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Definition
| facilitate the complex between the small ribosomal subunit, the mRNA and the special tRNAmet. |
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Term
| Two elongation factors, EFs, are needed for elongation. |
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Definition
| EF-T (pro)/EF-1 (euk) deliver charged tRNAs into the A site and insure the proper codon/anticodon recognition by hydrolyzing GTP. |
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Term
| Once the peptide bond forms between pepidyl-tRNA in the P site and the amino acyl-tRNA in the A site |
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Definition
| EF-G (pro)/EF-2 (euk) move the mRNA and tRNAs, hydrolyzing GTP in the process. |
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Term
| Initiation factors, proteins not bound to the ribosome, |
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Definition
| facilitate the complex between the small ribosomal subunit, the mRNA and the special tRNAmet. |
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Term
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Definition
|
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Term
| All RNAs have a leader sequence before the start codon. Ribosomes find the proper start codon either by |
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Definition
| base pairing between mRNA and rRNA (prokaryotes) or by scanning from the capped 5’-end (eukaryotes) |
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Term
| Peptide bonds (amide bonds) form from the |
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Definition
| nucleophilic attack of the amino group on the amino acid held by the tRNA in the A site to the ester linkage between the growing peptide attached to the tRNA in the P site. Thus, the peptide, elongated by one residue, is in the A site. |
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Term
| No direct expenditure of energy occurs during peptide bond formation. The enzyme that catalyzes the reaction |
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Definition
| (peptidyl transferase) is the rRNA of the large ribosomal subunit. This is an example of a ribozyme. |
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Term
| Release factors hydrolyze the ester linkage between |
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Definition
| the C terminal amino acid and the final tRNA. |
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Term
| Release factors hydrolyze the ester linkage between |
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Definition
| the C terminal amino acid and the final tRNA. |
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Term
| Release factors hydrolyze the ester linkage between |
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Definition
| the C terminal amino acid and the final tRNA. |
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Term
| Polysomes are mRNAs that have |
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Definition
| more than one ribosome carrying out translation |
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Term
| Many antibiotics made by eukaryotic microorganisms |
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Definition
| disrupt bacterial translation |
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