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BSCI330 > Lecture 13: Control of Gene Expression Part 1 - Regulation of mRNA levels > Flashcards

Lecture 13: Control of Gene Expression Part 1 - Regulation of mRNA levels Flashcards

1
Q

Why is gene expression regulated at essentially every step from transcription through protein activity/stability?

A
  • Can control how long mRNA stays in cytosol
  • Can control how ribosome interacts with mRNA
  • Can control whether a protein is active or inactive
  • This regulation puts in the exact amount of energy needed in order for proteins to be ready when needed
  • Transcriptional control can match the amount of mRNA to the amount of protein needed (if the protein is needed don’t bother making it)
  • Protein activity control can allows proteins that are needed immediately after cell recognition to become activated right away
  • Allows the cell to respond in varying levels to signal the most efficient way possible
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2
Q

Regulation of RNA transcription

A
  • For most genes, the primary regulation occurs at the level of RNA transcription
  • > Matching RNA synthesis to expression requirements avoids the expense of synthesizing unneeded macromolecules (remember how energetically costly that can be)
  • Sequence-specific DNA binding proteins, called gene regulatory proteins or transcription factors, play a key role in defining the level of transcription
  • > Transcription factors generally contain one or more of a small set of well-characterized DNA-binding motifs
  • Transcription factors can bind to, and read, the outside of the DNA helix and influence the binding or activity of RNA polymerase II
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3
Q

Why is the major groove the site of binding?

A
  • For major groove and minor groove, reading molecule can only see combinations of positively charged and negatively charged atoms
  • Major groove has more specific combinations so it’s possible to distinguish the order of nucleotides
  • The major groove presents a unique signature for each base pair
  • Each base pair has a pattern that can distinguish between the different base pairs (G-C vs. C-G and A-T vs. T-A)
  • The base pairs in the major groove are asymmetrical, so it’s easier to tell the difference unlike in the minor groove
  • Since it’s easier to distinguish the different base pairs, the DNA-binding motifs bind to the major groove
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4
Q

A DNA-binding protein can interact with specific base pairs without unzipping DNA

A
  • Interactions between the gene regulatory protein amino acid side chains and a base-pair can occur through hydrogen-bonding
  • Typically 10-20 contacts are made by a gene regulatory protein with DNA, so DNA-binding proteins usually recognize a whole sequence
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5
Q

Helix-turn-helix

A
  • One of the simplest DNA-binding motifs
  • Two alpha helices connected by a short unstructured stretch (“turn”)
  • Helices are held at a specific angle by interactions between the helices
  • C-terminal recognition helix makes sequence-specific contacts in the major groove of DNA
  • Generally bind to DNA as symmetric dimers, where recognition helices bind to “half-sites” separated by one turn of the DNA helix
  • This means that they’ll be interacting on the same side of DNA
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6
Q

Homeodomain

A
  • A special case of helix-turn-helix motif
  • A larger structure that includes a helix-turn-helix region plus other highly conserved structures (including a third alpha helix)
  • Conserved structure suggest that all homeodomains are presented to DNA in the same fashion
  • More extensive contacts with the DNA, as 2 of the alpha helices make contact with the DNA
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7
Q

Zinc fingers first subclass

A
  • One or more zinc ions is coordinated by amino acid side groups
  • One subclass uses 2 cysteines and 2 histidines to coordinate zinc between an alpha helix and a 2-strand antiparallel beta sheet
  • Often found in tandem clusters within a DNA-binding protein (longer DNA sequences get recognized)
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8
Q

Zinc fingers second subclass

A
  • Second subclass coordinates 2 zinc ions, using 4 cysteines for each
  • Two regions: one zinc ion stabilizes a recognition helix and one stabilizes a loop involved in dimerization
  • Bind to DNA as symmetric dimers, similar to helix-turn-helix proteins
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9
Q

Leucine zipper

A
  • One long alpha helix containing a hydrophobic surface on one side and a hydrophilic surface on the other
  • Protein binds DNA as a dimeric structure
  • The helix from one subunit binds to the corresponding helix in the second subunit in a coiled-coil structure - hydrophobic interactions
  • The hydrophobic surface on the alpha helix has large hydrophobic amino acids sticking out, and often they are leucines/iso-leucines and they will interact with the amino acids on the other subunits along the alpha helix, giving it a zipper structure
  • The long alpha helix serves both as the dimerization region and the DNA-binding region
  • The subunits are binding a half-turn apart, not a full turn apart
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10
Q

Helix-loop-helix (HLH)

A
  • Not the same as helix-turn-helix
  • A short alpha helix is connected to a longer alpha helix by a flexible loop
  • Loop allows one helix to fold back and pack against the other
  • As with the leucine zipper, the HLH motif acts as both a dimerization interface and the DNA-binding
  • Subunits are not a full turn of DNA apart
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11
Q

What does dimerization of DNA-binding proteins do?

A
  • Can enhance binding (make binding stronger) and specificity by increasing the contact area with DNA
  • The longer the DNA sequence that needs to be recognized, the less likely it’ll be in the DNA randomly and more likely to be in specific areas of the DNA
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12
Q

What does heterodimerization do?

A
  • Between 2 different members of the same class
  • Increases the range of sequences that can be recognized - power of combinatorial math
  • For example, 2 different leucine zippers
  • This allows more DNA sequences to be recognized without increasing the number of proteins in the cell
  • The more different the heterodimers, the more DNA sequences that can be recognized
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13
Q

How is RNA transcription regulated?

A

Transcription factors generally act out one of two types of gene regulatory regions: promoter or enhancer

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14
Q

Promoter

A
  • The region where RNA polymerase and the general transcription factors assemble
  • It’s always a short distance “upstream” of the 5’ end of the gene
  • This region is absolutely required for transcription initiation, but may only provide a low level of transcription
  • May be gene-specific (i.e. not work if it’s put next to a different gene, and its orientation may be important
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15
Q

Enhancer

A
  • An independent region outside the promoter
  • It may be very far away from the promoter (up to 10s of kb) and may be upstream of the gene, downstream, or even within the gene
  • This region cannot drive transcription on its own, but dramatically increases transcription initiation from its corresponding promoter
  • Are generally position- and orientation-independent, and can work with a heterologous promoter (i.e. the promoter from a different gene)
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16
Q

How do Transcription factors work?

A
  • Eukaryotic gene regulatory region are typically much more complex than prokaryotic regulatory regions
  • Multiple (sometimes dozens or hundreds) of gene regulatory proteins work together to control the role of transcription - combinatorial control of expression
  • TF’s may work cooperatively (e.g. two activators) or antagonistically (e.g. activator vs. a repressor)
  • The combination of different regulatory factors allows us to fine tune expression
  • Cooperative interactions may increase transcription synergistically (more than simply additive effects)
  • Proteins can regulate distant genes by DNA folding so they can interact with a large transcription complex
  • Combining proteins into a larger structure increases the structure’s stability so more molecules of RNA can be made
  • Transcription factors can serve as activators or repressors
  • > Many transcription factors are activators
  • Transcription factors can direct local alterations in chromatin structure
17
Q

What mechanisms do transcription factors use to regulate gene expression?

A
  • Some help to unpack or pack chromatin, making the gene accessible to RNA polymerase and the initiation complex
  • Some control recruitment of RNA polymerase and/or the general transcription factors to the promoter
  • Some may regulate the switch from initiation to elongation
  • Some help recruit histone-modifying enzymes to change the local chromatin structure (local promoter change)
  • Some bend DNA to allow long-distance interactions between gene regulatory regions
18
Q

How are Transcription factors regulated?

A
  • Must be selectively activated - cannot have every one turned on in every cell at all times
  • Many are themselves regulated at the levels of gene transcription
  • > Tissue-specific expression (present in liver, but not in lymphocytes)
  • > Expressed only in response to specific environmental signals (cell starves)
  • > Expressed during specific phases of cell cycle (on during mitosis but not during G1)
  • Many are present in an inactive state, and are activated by phosphorylation
  • > The mitogen activated protein kinase family is important for phosphorylating a variety of transcription factors in response to signals from all-surface receptors
  • > Phosphorylation converts an inactive form into an active form, or vice versa
  • Transcription factors often have multiple sites for phosphorylation and other modifications - molecular integrators
19
Q

How can the transcription of transcription factor genes be selectively regulated?

A
  • This remains a chicken and eggs problem, though
  • Some transcription factors must be regulated by other mechanisms
  • If not regulated at level of transcription, must be regulated post-transcriptionally
  • p53 is modified at over 20 different sites within its structure
  • > Each modification site can respond to different signals and have slightly different effects on the transcription activity of p53
  • > Allows p53 to recognize a lot of different signals and integrate them into a single output
20
Q

Post-transcriptional regulation of transcription factors

A
  • Post-translational modification may not regulate transcription factors activity directly, but instead acts by changing the cellular localization
  • Variations on a theme: NF-AT and NF-kB
  • > Held in the cytosol in an “inactive” state
  • > Post-translational modifications lead to release from the cytosol and translocation to the nucleus
  • > Nuclear transcription factor then is able to regulate gene transcription
  • Many transcription factors are regulated by a combination of expression, activation, and localization
  • > In order to get exact level of activity for certain conditions
21
Q

Combinatorial control can generate patterns during animal development

A
  • E.g. Even-skipped (Eve) expression in fly embryo
  • Expression in one stripe is directed by one DNA molecule
  • > If a stripe module is placed upstream of a basil promoter, that module only regulates expression in that specific stripe
  • > Each module works independently
  • Combination of gene regulatory proteins that bind to this DNA module dictate expression
  • > Example: stripe 2 module contains binding sites different activator and repressor genes that dictate the expression of genes within the stripe 2 module
  • > The combination of the binding sites dictates whether a transcription factor is expressed or not expressed in stripe 2
  • Patterns of expression of these gene regulatory proteins makes the right combination available only in one stripe
  • Combination of gene regulatory proteins that bind to this DNA module dictate expression
  • > Gives a very specific pattern of expression
  • Similar combinatorial logic may regulate the expression of globin genes in mammals
22
Q

More Transcription regulation

A
  • RNA levels cn be regulated at the level of initiation or termination
  • Transcription attenuation leads to premature termination of the RNA transcript
  • > The growing RNA chain adopts a conformation that interferes with RNA polymerase activity
  • > RNA polymerase pauses, and eventually aborts transcription
  • > Attenuation can be reversed by binding of specific proteins to the RNA structure, allowing RNA polymerase to complete transcription
23
Q

Barrier sequences

A
  • Binds proteins that inhibit the spread of heterochromatin and/or tether the DNA to the nuclear membrane
  • Prevents spurious spread of transcriptional control
24
Q

Insulator elements

A
  • May be decoys that tie up the transcription machinery or may tether the DNA to the nuclear membrane to prevent DNA looping
  • Prevents spurious spread of transcriptional control

BSCI330 (12 decks)

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