CS&B - Biochemistry - Central Dogma; Signaling; Recombination Flashcards
What is the central dogma?
DNA — (transcription) —> RNA — (translation) —> Protein
Addition of the _________ cap to the 5’ end of hnRNA and a poly-A tail to the 3’ end are both associated with what portion of the RNA polymerase II that coded the hnRNA strand?
7-methylguanosine;
the carboxy-terminal domain (CTD)
What is the name for the raw RNA strand transcribed from DNA?
This is modified to become what?
hnRNA;
mRNA
What must occur for hnRNA to become mRNA?
5’ cap;
3’ poly-A tail;
splicing out of of introns
___________ are made of various proteins including snRNPs.
Spliceosomes
Via what shape do spliceosomes remove hnRNA introns?
Lariats
What molecule provides the methyl group to 7-MeGuanosine?
This molecule is replenished by what vitamins?
S-adenosylmethionine;
folate, B12 (via methionine)
What enzyme adds the poly-A tail to the 3’ end of an hnRNA?
What nucleotide sequence does it follow?
Poly-A polymerase;
AAUAAA
Will nucleopores allow hnRNA to leave the nucleus?
Will nucleopores allow mRNA to leave the nucleus?
No;
yes
What are the two portions of a eukaryotic ribosome?
Which binds to the mRNA first?
60s, 40s;
40s
What proteins are responsible for connecting the 40s ribosomal subunit to an mRNA strand?
What is their energy source?
Eukaryotic initiation factors (eIFs);
GTP
What are the four main eukaryotic initiation factors (eIFs)?
What is their overall goal?
eIF2, eIF3, eIF4a, eIF4b;
unite the 40s ribosomal subunit with an mRNA
What does the eIF2 (eukaryotic initiation factor 2) do?
What does the eIF3 (eukaryotic initiation factor 3) do?
What does the eIF4a (eukaryotic initiation factor 4a) do?
What does the eIF4b (eukaryotic initiation factor 4b) do?
Facilitates 40s ribosomal subunit binding;
binds 40s;
removes 2° mRNA structures (it is an RNA helicase);
locates AUG
What does the eIF2 (eukaryotic initiation factor 2) do?
Facilitates 40s ribosomal subunit binding
What does the eIF3 (eukaryotic initiation factor 3) do?
Binds 40s
What does the eIF4a (eukaryotic initiation factor 4a) do?
Removes 2° mRNA structures (it is an RNA helicase)
What does the eIF4b (eukaryotic initiation factor 4b) do?
Locates AUG
After reaching the end codon (UGA, UAA, or UAG), what protein family remove the 40s and 60s ribosomal subunits from the mRNA strand?
Eukaryotic release factors (eRFs)
Which end of a tRNA holds the amino acid?
The 5’ end
The initial tRNA holds what amino acid?
On which end?
Methionine;
5’
In what order of binding sites does a tRNA pass through the ribosome?
A –> P –> E
What is the A site of the ribosome?
Where the aminoacyl-tRNA enters the ribosome.
What is the P site of the ribosome? What two things happen here?
Where the aminoacyl-tRNA anticodon binds the mRNA codon;
the amino acid is joined to the growing chain via peptidyl transferase
What is the E site of the ribosome?
The exit point for the empty tRNA
What proteins bind recently translated mRNA to protect them and help them fold?
Chaperone proteins
(e.g. heat-shock proteins)
Proteins can be heavily phosphated and then tagged with what marker to target them for degradation?
What is this process called?
What enzyme attaches the marker?
Ub;
ubiquination;
ub-ligase
What complex degrades ubiquinated proteins?
The 26S proteasome
What is a silent mutation?
What is a frameshift mutation?
What is a nonsense mutation?
No change in coded amino acid (often due to RNA wobble);
Insertion or deletion (change in reading frame);
premature stop codon (truncated protein)
What are the stop codons?
For what amino acid do they code?
What is the start codon?
For what amino acid does it code?
UAA, UAG, UGA,
none;
AUG,
methionine
The wobble hypothesis involves which of the three nucleotides of an RNA codon?
The third
What enzyme normally cleaves APP extracellularly?
What enzyme normally cleaves APP intracellularly?
What enzyme is pathological in its extracellular cleavage of APP?
Alpha-secretase;
gamma-secretase (PSEN1);
beta-secretase
What form of G-protein is associated with G-protein coupled receptors?
Trimeric
(as opposed to monomeric)
Describe the general structure of a GPCR.
One receptor (7 membrane-spanning domains) connected to an intracellular, trimeric G-protein
(Ligand + receptor + G-protein + effector enzyme = induced action)
G-proteins hydrolyze what substrate for energy?
GTP
What are the three subunits of the G-protein in a GPCR?
Gα, Gβ, Gγ
Which of the G-protein trimeric subunits are anchored to the P-leaflet of the plasma membrane?
Gα, Gγ
What does the Gα subunit of a GPCR do?
Binds GDP (or GTP);
modifies the effector molecule (e.g. adenylyl cyclase)
After a ligand binds the receptor portion of a G-protein coupled receptor, what occurs?
The Gα subunit exchanges GDP for GTP,
dissociates from the GPCR,
and binds to an effector molecule
Upon ligand binding, which of the trimeric subunit of a GPCR dissociates from the GPCR and binds to an effector molecule?
What subunits are there?
Gα;
Gα, Gβ, Gγ
To what is the Gα subunit bound when a ligand binds the GPCR receptor and what occurs next?
GDP;
the GDP is exchanged for GTP;
the Gα dissociates from the GPCR
After leaving the GPCR and binding the effector molecule, how does the GTP-bound Gα dissociate from the effector to return to the GPCR?
It hydrolyzes the GTP
True/False.
A single effector molecule (such as adenylyl cyclase) can have both stimulatory and inhibitory GPCRs.
True.
Describe the pathway of a GPCR that acts on adenylyl cyclase.
Ligand binds GPCR –>
Gα (trades GDP for GTP) dissociates –>
binds adenylyl cyclase –>
creates cAMP –>
activates protein kinase A
What secondary messenger is created by adenylyl cyclase?
What enzyme does it activate?
cAMP;
protein kinase A (PKA)
Describe the pathway of a GPCR that acts on guanylyl cyclase.
Ligand binds GPCR –>
Gα (trades GDP for GTP) dissociates –>
binds guanylyl cyclase –>
creates cGMP –>
activates protein kinase G
What secondary messenger is created by guanylyl cyclase?
What enzyme does it activate?
cGMP;
protein kinase G (PKG)
Describe the pathway of a GPCR that acts on phospholipase C.
Ligand binds GPCR –>
Gα (trades GDP for GTP) dissociates –>
binds phospholipase C –>
creates inisitol triphosphate (IP3) –> releases stored Ca2+
and diacylglycerol (DAG) –> activates protein kinase C
What secondary messengers are created by phospholipase C?
What substances do they activate and release, respectively?
Diacylglycerol (DAG) –> protein kinase C (PKC);
inisitol triphosphate (IP3) –> Ca2+
What type of adrenergic input increases adenylyl cyclase activity? Through what Gα subunit?
What type of adrenergic input decreases adenylyl cyclase activity? Through what Gα subunit?
Beta-adrenergic, Gαs (turn on the gas)
Alpha-adrenergic, Gαi (stop that guy)
What secondary messenger is elevated in cases of adenylyl cyclase activation?
cAMP
What secondary messenger is elevated in cases of guanylyl cyclase activation?
cGMP
What secondary messengers are elevated in cases of phospholipase C activation?
diacylglycerol (DAG),
inisitol triphosphate (IP3),
Ca2+
How does cAMP exert an effect on protein kinase A?
It binds the 2 regulatory subunits,
these activate the 2 catalytic subunits,
these enter the nucleus
After being activated by cAMP, where do the catalytic PKA subunits go and what effect do they have?
Into the nucleus;
they phosphorylate the CREB protein,
which binds CRE DNA consensus sequences
Besides affecting gene transcription through its catalytic units, what does cAMP-activated PKA do to affect gene transcription through alternate kinase activity?
Phosphorylates the mitogen-activated protein kinase (MAPK)
What sorts of extracellular signals will activate GPCRs that eventually activate PKA?
What are some of the effects in adipose tissue, liver tissue, and skeletal muscle?
Norepinephrine, epinephrine, ACTH, glucagon;
increased serum fatty acids, amino acids, and glucose
(increased lipolysis, decreased glycogenolysis, increased glycogenesis, decreased amino acid uptake, increased gluconeogenesis etc.)
Will cAMP-PKA signaling have the same effect on each tissue in which it is utilized?
No
(liver responses are very different from ovarian responses which are very different from kidney responses, etc.)
Phospholipase C acts on what plasma membrane substrate to produce DAG and IP3?
PIP2
(phosphatidylinositol + 4-,5-phosphates)
(PI from the P-leaflet)
After phospholipase C acts on PIP2 to form DAG and IP3, what happens next?
IP3 triggers release of Ca2+ from the ER;
Ca2+ then helps PKC to go out towards the plasma membrane where DAG activates it
Smooth muscle relaxation is a GPCR-stimulated response that is an example of what sort of cell-to-cell signaling?
Paracrine signaling
Acetylcholine binds a GPCR on a smooth muscle cell. What effector enzyme is affected and how?
What is the effect?
Phospholipase C, activated;
increased Ca2+ binds calmodulin and activate nitric oxide synthase
(which diffuses and thus relaxes surrounding myocytes)
After acetylcholine binds a smooth muscle GPCR, phospholipase C is activated and calcium released from the sarcoplasmic reticulum.
This calcium then binds to what?
The union of calcium and this other molecule then have what effect?
This nitric oxide then does what?
Calmodulin;
activate nitric oxide synthase;
binds a GPCR for guanylyl cyclase (causing smooth muscle relaxation)
Nitric oxide binds to what receptor on smooth muscle cells?
GPCR –> guanylyl cyclase –> cGMP –> PKG
Phospholipase C activation has what effect on smooth muscle cells?
Guanylyl cyclase activation has what effect on smooth muscle cells?
Nitric oxide production and subsequent guanylyl cyclase activation;
relaxation and blood vessel dilation
When is the Gα subunit (of a GCPR) active?
When bound to GTP
What are the three main categories of single pass receptor signaling pathways?
(C, RTK, T)
Cytokine receptors;
receptor tyrosine kinase;
TGFβ receptors
What type of single pass receptor is responsible for JAK/STAT signalling?
Cytokine receptors
Do cytokine receptors have kinases that are intrinsic (part of) or extrinsic (just nearby or associated) to the receptor itself?
Extrinsic (the JAK kinase)
What is the name of the kinase that is extrinsic and closely associated with cytokine receptors?
JAK
When a ligand binds to a cytokine receptor monomer, what happens?
Two monomers are brought together, and their extrinsic (but intracellular) kinases are united
When cytokine receptors dimerize (upon meeting a ligand) and their extrinsic JAK kinases come together, what happens?
The JAK kinases phosphorylate and activate one another;
they then phosphorylate tyrosine residues on the intracellular portion of the receptor itself
Why is it important that JAK kinases phosphorylate the intracellular portions of activated cytokine receptors?
So, the phosphorylated proteins can serve as scaffolding/cascade for signalling proteins
What is the STAT (of the JAK/STAT cytokine receptor pathway) part of JAK/STAT?
A transcription factor
(SH2 + a DNA-binding domain)
What is the JAK (of the JAK/STAT cytokine receptor pathway) part of JAK/STAT?
The kinase associated with cytokine receptors
What are some examples of substances that interact with cytokine receptors?
Interferons,
prolactin,
EPO
Cytokine receptors and the JAK/STAT pathway are typically pro- what?
Pro-growth
(and sometimes differentiation)
Besides being pro-growth, what other effect does the JAK/STAT pathway of cytokine receptors have on cell responses to noxious stimuli?
It is anti-inflammatory
The eukaryotic initiation factor (eIFs) “eIF4B” is specifically responsible for:
Rhe scanning of mRNA to locate the start codon “AUG”
After transcription termination, ___________ cleave the hnRNA near the ______ signal, and poly-A polymerase adds the tail.
Endonucleases, poly-A
Via which G-protein is a signal transferred to the effector adenylyl cyclase to stimulate increased cAMP production?
Gα stimulatory (Gαs) subunit
(turn on the Gαs)
Often, single pass receptors (cytokine receptors, RTKs, TGFβ receptors) exist as single ___________, but, after ligand binding, unite to become ____________.
Monomers;
dimers
(Image: example cytokine receptor)
Describe the pathway of a receptor tyrosine kinase.
- Ligand binding and receptor dimerization
- Kinase activation (of each other)
- Receptor phosphorylation; signal cascade activation
Which has an extrinsic kinase, receptor tyrosine kinases or cytokine receptors?
Cytokine receptors
(extrinsic = associated but not directly part of the receptor)
GPCRs use _____________ G-proteins.
Many single pass receptor pathways use ______________ G-proteins.
Trimeric;
monomeric
A monomeric G-protein is active if it is bound to:
A monomeric G-protein is inactive if it is bound to:
GTP;
GDP
A monomeric G-protein is active if it is bound to GTP.
What activating enzyme exchanges G-protein GDP for GTP?
A monomeric G-protein is inactive if it is bound to GDP.
What inactivating enzyme exchanges G-protein GDP for GTP?
GEF
(guanine nucleotide exchange factor);
GAP
(GTPase activating protein)
What enzyme activates monomeric G-proteins?
Through what mechanism?
GEF (guanine nucleotide exchange factor);
trading G-protein GDP for GTP
What enzyme inactivates monomeric G-proteins?
Through what mechanism?
GAP (GTPase activating protein);
promoting the hydrolysis of G-protein GTP to GDP
The Sos protein is activated by receptor tyrosine kinases to do what to what G-protein?
Activate (Sos is a GEF);
Ras
Ras is a __________ and is part of what signalling pathway?
JAK is a __________ and is part of what signalling pathway?
Monomeric G-protein, receptor tyrosine kinase;
kinase, cytokine receptor
After a ligand binds receptor tyrosine kinase, what is the cascade order of protein activations?
Sos (a GEF, a GDP to GTP exchanger) activates Ras;
Ras –> Ref –> MEK –> MAPK (ERK)
What mutation in the receptor tyrosine kinase pathway leads to overexpression of MAP kinase activity and increased likelihood of cancer development?
Ras (a monomeric G-protein) mutates and is perpetually active
What type of ligands bind to receptor tyrosine kinases?
Insulin,
growth factors (e.g. EGFR or VEGFR)
What is the basic signalling pathway of a TGFβ receptor?
- TGFβ binds receptors II and III, receptor I is then recruited
- The receptor intrinsic kinases activate one another
- SMAD3 and SMAD4 combine and are chaperoned to the nucleus
- SMAD3-SMAD4 alter gene transcription
TGFβ signalling causes modified gene transcription with what two main effects regarding the extracellular matrix?
- Increased ECM secretion
- Secretion of plasmogen activator inhibitor 1 (PAI1)
(basically, more extracellular protein fibers and less degradation of the fibers –> more stable tissues)
TGFβ signalling causes modified gene transcription with what main effect regarding cellular growth?
TGFβ signalling is anti-growth
(9CDKI p15 and PAI1 expression)
Inactivating mutations in the TGFβ signalling pathway would have what effect?
Excess growth and increased cancer risk
(e.g. SMAD or receptor I or II mutations in RB and cancers of the colon, pancreas, stomach)
An inactivating mutation in the SMAD4 protein or the receptor I or II would have what effect on cellular activity?
Increased growth and cancer risk
What is an example of a serum protein with the opposite effect of tissue plasminogen activator (TPA)?
Plasminogen activator inhibitor 1 (PAI1)
Oncogenic mutations involving SMAD4 are related to what single pass transmembrane receptor pathways?
Oncogenic mutations involving Ras are related to what single pass transmembrane receptor pathways?
Mutations involving JAK are related to what single pass transmembrane receptor pathways?
TGFβ receptors;
receptor tyrosine kinases;
cytokine receptors
What is the purpose of DNA recombination?
Mixing and rearranging the genome
(creates more genetic diversity and variability among humans)
How many principal types of recombination exist?
What are they?
3;
homologous r.,
site-specific r.,
transposition
When does homologous recombination occur?
What are its purposes?
Gametogenesis,
somatic replication;
increase genetic diversity, DNA repair
When does site-specific recombination occur?
What is one way that it is medically relevant?
Mainly studied as specific exchanges between bacteria and bacteriophages;
antibiotic resistance
Through what type of DNA sequence does transposable recombination occur?
What does it accomplish?
Transposons (up to 50% of genome)
antibody and genetic diversity
What is another term for homologous recombination?
What is the term for the point of DNA ‘swapping?’
What enzymes are involved?
Crossing over;
chiasma;
the RecBCD complex and RecA enzyme
What is here described: reciprocal chiasmatic exchange of genetic information between homologous chromosomes during gametogenesis.
Homologous recombination
True/False.
Homologous recombination sometimes leads to tumor formation.
True.
When during the cell cycle does homologous recombination occur in a gamete?
Prophase I
Homologous recombination can be used to map what on a chromosome?
Genetic loci
True/False.
The further apart two loci are, the more likely it is that recombination will occur.
True.
Homologous recombination can be used to ‘skip’ past what type of DNA error?
One that stalls replication
(e. g. a thymidine dimer)
* (Note: in lefthand portion of image, pay attention to the location of the lesion in each step)*
How can homologous recombination help DNA replication to ‘skip’ past large DNA lesions such as thymidine dimers?
By regressing and using the newly synthesized strand opposite it as a template
(Note: in lefthand portion of image, pay attention to the location of the lesion in each step)
Homologous recombination can be used to repair small DNA nicks by uniting what two strands?
The leading and lagging strands
(Note: righthand portion of image)
What is the term for chromosomes where no crossing over has occurred?
What is the term for chromosomes where crossing over has occurred?
Parental;
recombinant
Homologous recombination (crossing over) can have what effect on an individual heterozygous for a particular allele?
Is this true for meiosis or mitosis?
Loss of heterozygosity
(the alleles replicate for mitosis [2 –> 4], and then the gene is swapped so each daughter cell is homozygous for one of the original two alleles);
mitosis
True/False.
Mitotic (or meiotic) nondisjunction is a form of homologous recombination.
False.
What type of recombination requires shared DNA sequences in order to swap genes between a bacterium and a bacteriophage?
Site-specific recombination
What sequences border transposable sequences?
What sequences border the target location of transposition?
Terminal repeats
(for easy endonuclease activity, I assume);
duplicated flanking sequences
The ________ _________ on either end of a transposon bind the __________ ____________ sequences of the target insertion site.
Terminal repeats;
duplicated flanking
What category of transposons are ‘copy-and-pasted’ around the genome?
What category of transposons are ‘cut-and-pasted’ around the genome?
Retrotransposons;
DNA transposons
What are the two categories of retrotransposons (copy and paste transposons)?
Interspersed nuclear elements (short and long)
–> LINEs and SINEs (SINEs = most common)
retroviral LTRs (long terminal repeats)
Are many DNA transposons inactive?
Yes;
nearly all
How are retrotransposons ‘copy and pasted’ throughout the genome?
RNA polymerase makes an RNA template;
reverse transcriptase then turns it back into DNA
(much like a retrovirus)
How are DNA transposons ‘cut and pasted’ throughout the genome?
Endonucleases and ligases
Excision of various transposons is essential to the diverse variability of what type of cellular immune product?
Antibodies
What are the three segments of antibody light-chain DNA sequences?
V segments (many which can be swapped in and out);
J segments (a few, out of which one type is usually selected);
C (the constant portion)
What sequences and enzymes are responsible for this process of antibody light-chain transposon recombination?
Recombination signal sequences (RSSs);
RAG1, RAG2
What is ‘exon shuffling?’
Switching of exons among various genes
(transposition recombination)
How can the frequency with which two genes are swapped in homologous recombination be useful to mapping out genetic loci on a chromosome?
The farther apart two genes are, the more likely it is that there will be some crossing over between them
(the frequency is proportional to the distance between the loci)
Can multiple eukaryotic ribosomes be actively translating a single mRNA strand simultaneously?
Yes
(polyribosomal activity)
What post-translational protein modifier converts proline from the trans configuration to the cis configuration?
What post-translational protein modifier creates disulfide bonds between cysteine residues?
Note: These enzymes, along with chaperones and HSPs, are both important to proper protein folding.
Peptidyl prolyl cis-trans isomerase;
protein disulfide isomerase
What is the shared purpose of the following three mechanisms:
glycosylation of phosphatidylinositol,
acetylation,
prenylation?
Anchoring proteins to the lipid membrane
What is added to a substrate during carboxylation?
A carboxyl (COO-) group
What is the term for an inactive enzyme that is lacking its specific prosthetic group?
What is the term for an active enzyme that is accompanied by its specific prosthetic group?
Apoprotein, apozyme;
holoenzyme
Which of the following signal types is(are) generally pro-growth?
Which of the following signal types is(are) generally anti-growth?
Cytokine receptors
Receptor tyrosine kinases
TGFβ receptors
Cytokine receptors,
Receptor tyrosine kinases;
TGFβ receptors
