BIO Exam 4

What sequences are at the edges of introns? What do they do?

5' end = GU
3' end = AG
Polypyrimidine tract - before the 3' end AG

Indicate where the intron begins and ends

What is the spliceosome? What is it made of?

A large complex consisting of protein AND RNA that catalyzes the splicing reaction

What are snRNP's made of?

snRNA [small nuclear RNA] and protein

What does U1 snRNP do?

Binds to 5' splice site

What does U2 snRNP do?

Binds to the branch point (Adenine)

What do U3/U4/U5 snRNP's do?

Brings the two ends of the excised intron lariat complex together by associating U1 and U2

What is a lariat?

An circular, excised intron

Spliceosomes are assembled from a group of smaller RNA-protein complexes called...

snRNP's

What protein is added where two exons come together?

Exon Junction Complex

What does the Exon Junction Complex do?

1. Signals the RNA to leave the nucleus
2. Tells us if the RNA was spliced wrong (nonsense) and signals for the RNA to be degraded

What is alternative splicing?

The order of exons are rearranged by cutting out multiple introns, including the exons between them

How does RNA processing occur cotranscriptionally?

Many phosphorylated repeats of a seven amino-acid sequence on the tail of the RNA polymerase bind to enzymes needed for capping, splicing, and cleavage.

What is nuclear RNA export factor 1? What is its function?

A protein which interacts with the cap and the exon junction complex which signals that an mRNA transcript is ready for export from the nucleus.

What are the 5 components required by translation?

ribosomes, tRNA, aminoacyl-tRNA synthetase, mRNA, protein factors

What is the structure of ribosomes?

Made of rRNA, with a large and small subunits.

What are the ribosomal subunits in eukaryotes vs bacteria?

In bacteria, 50S/30S. In eukaryotes 60S/40S

What are the four important sites on the ribosome? Where is each site located?

1. mRNA binding site (part of small subunit)
2. A amino acid site (large subunit)
3. P peptide site (large subunit)
4. E exit site (large subunit)

What is the function of the anticodon?

Part of tRNA which base pairs with the codon

What is the function of the 3' CCA in tRNA?

Binding site for the amino acid

A charged tRNA has...

an amino acid attached to it

What is wobbling?

Genetic code is redundant- multiple codons code for the same amino acid. The bond between the tRNA and the third base of the codon is the weakest.

The ___ base of a codon is the least effected by mutation

third

Which is the wobbliest base?

Inosine

What is the function of aminoacyl tRNA synthetase?

Uses ATP's conversion to AMP to link an amino acid to a tRNA molecule. Also proofreads that the proper amino acid has been added to the matching tRNA.

What is the start codon? What amino acid does it carry?

AUG. Methionine.

What are the stop codons?

UAA, UAG, UGA

What are UTR's?

Untranslated regions of mRNA

What are the three stages of translation?

Initiation, elongation, termination

Proteins grow from the ____ to the ____

N terminus to the C terminus

Which subunit binds first during initiation of translation? Why?

The small subunit, because it has the mRNA binding site.

What is a Shine-Delgarno sequence?

Sequence on PROKARYOTIC mRNA which serves as a binding site for the small ribosomal subunit

What three initiation factors are found in initiation of translation in Bacteria? What are their functions?

IF1 - carries IF2
IF2 - carries GTP which helps things bind
IF3 - Blocks the large subunit from binding to allow the tRNA to bind first.

What is the difference between initiation of translation in eukaryotes vs bacteria?

1. In eukaryotes, 12 initiation factors instead of 3
2. In eukaryotes, Methionine is not going to be formulated
3. In eukaryotes, No Shine-Delgarno sequence

Initiation factors that start with a 4 are involved in...

binding to the 5' cap

What are the 3 steps of elongation?

1. Binding of aminoacyl tRNA to the ribosome
2. Peptide bond formation
3. Translocation

What proteins facilitate termination of translation? How do they work?

Release factors bind to the A site of the large ribosomal subunit, dissociating the ribosome from the mRNA.

What is a Kozak sequence?

Site for initiation of translation in EUKARYOTES.

What sequences do release factors recognize in termination of translation?

The stop codons

Antibiotic mechanisms tend to interfere with ___

protein translation

What are molecular chaperones?

Proteins which facilitate the polypeptide folding process

What are the two most widely occurring molecular chaperone families?

Hsp70, Hsp60

How does Hsp70 work?

Multiple Hsp70 proteins (and associated proteins) attach to a polypeptide using ATP. Once properly folded, the Hsp70 proteins detach.

How does Hsp60 work?

An unfolded polypeptide enters the GroEL subunit. A GroES subunit attaches to one end (with hydrolysis of 7 ATP molecules), causing GroEL to change shape and creating a hydrophilic environment that leads to protein folding. Once folded, another GroES attaches to the other side and the protein is released.

How many chances does a polypeptide get to fold?

two

What protein is used to mark other proteins for destruction?

Ubiquitin

Ubiquitin is attached to...

lysine residues

Proteins marked for destruction are destroyed in a...

proteasome

Marking proteins for destruction requires ATP hydrolysis (True/False)

True, attaching ubiquitin to E1 requires ATP hydrolysis

Breaking down proteins in proteasomes requires ATP hydrolysis (True/False)

True

What is a disease caused by a missense mutation? What amino acid(s) are changed?

Sickle cell anemia. A negatively charged glutamine is changed into a neutral valine.

What type of mutation occurs when a stop codon is altered to an amino acid codon?

A nonstop mutation

What is a missense mutation?

A base pair substitution that changes an amino acid

What is a nonsense mutation?

A base pair substitution that turns a codon into a stop codon

What is a silent mutation?

A base pair substitution that does not cause an amino acid change

What is a frameshift mutation?

A complete addition or deletion of a nucleotide

What is a suppressor tRNA? What does it do?

It is a mutated tRNA which can bind to a stop codon and put a random amino acid in its place.

How does the cell know if there is a premature stop codon?

When an exon junction complex is detected AFTER a stop codon.

What is the name for the process of destroying mRNA's with premature stop codons?

Nonsense-mediated decay

What groups might be removed during posttranslational processing?

a) N-formyl groups that were part of methionine (start codon) in bacteria
b) signal sequences and other amino acids
c) removal to activate inactive precursors

What are the forms of posttranslational processing?

removal of amino acids
chemical modification (phosphorylation/glycosylation/methylation)
assembling of multi subunit proteins
protein splicing

Where do proteins go in cotranslational import?

The endomembrane system

Where do proteins go in posttranslational import?

They are retained in the cytosol or transported to the mitochondria/chloroplast

What is the sequence found at the beginning of proteins in cotranslational import?

The signal sequence

What protein binds to a signal sequence in cotranslational import?

SRP (Signal Recognition Particle)

What are the characteristics of a signal sequence in cotranslational import?

- 15-30 amino acids long
- positively charged N-terminal end
- hydrophobic center
- contains a polar region

Describe the sequence of events in cotranslational import.

- SRP binds to the ER signal sequence on an emerging polypeptide
- SRP uses GTP to move the polypeptide into a pore on the rough ER membrane
- Polypeptide goes through the pore and into the ER lumen
- Signal peptidase cuts off the signal sequence and polypeptide begins folding

What is BiP?

A member of the Hsp 70 family that prevents polypeptide aggregates from forming by binding to exposed hydrophobic amino acids. It gives a protein time to refold and if not it triggers endoplasmic reticulum associated degradation (ERAD)

What is ERAD?

endoplasmic reticulum associated degradation

What is a stop-transfer sequence? What does it do?

An additional sequence added during cotranslational import which is hydrophobic and prevents the protein from fully entering the ER lumen. This embeds the protein in the ER membrane.

What is a start-transfer sequence? What does it do?

Found in proteins that do not have a signal sequence- this sequence binds to an SRP and is kept within the membrane during translation.

How are multi-pass transmembrane proteins integrated into the ER membrane?

Through multiple start and stop transfer sequences.

What is an N-terminal transit sequence?

A polypeptide sequence which binds to TOM/TOC and TIM/TIC complexes, causing the protein to be imported into the mitochondrial matrix

What are the long range messengers produced by a cell?

endocrine signals

Short range messaging between cells is called...

paracrine

What are the messengers sent between cells that require physical contact?

juxtacrine signals

What is a messenger that acts on the same cell that produces it?

an autocrine signal

What kind of bond is formed between a ligand and a receptor?

A highly specific bond with several noncovalent bonds

A high receptor affinity means...

It takes few ligands to fully occupy all of the receptors

A low receptor affinity means...

It takes a high concentration of ligand for most receptors to be occupied

What does the dissociation constant represent? (NOT the equation)

The concentration of free ligand needed to produce a state in which half of the receptors are occupied

What is an agonist? How is it different from an antagonist?

An agonist binds to a receptor and activates. An antagonist binds to a receptor and prevents it from being activated

What are the two ways in which signaling can be deactivated?

1) reducing the amount of free ligand
2) reducing the sensitivity of the receptor
3) changing the number of receptors present

What are coreceptors?

Molecules which interact with a receptor, facilitating its interaction with a ligand

What two outcomes are possible when a receptor binds to a ligand?

1) it could undergo a conformational change and/or
2) multiple receptors could cluster together

What are the four basic types of signalling pathways?

1. Ligand-gated ion channels
2) G protein-coupled receptors
3) Enzyme-coupled receptors
4) Nuclear receptors

The C-terminusend of a GPCR is in the...

cytosol

The N-terminus end of a GPCR is in the...

outside of the cell

When G protein coupled receptors are phosphorylated, they are...

desensitized

Where are G protein coupled receptors phosphorylated? What protein phosphorylates them?

cytosolic domain. G protein coupled receptor kinases.

Describe the Gs protein pathway

1) Ligand binds to a transmembrane receptor
2) G alpha binds to GTP and seperates from beta/gamma
3) G alpha binds to a target protein like adenlyl cyclase
4) cAMP is produced and activates target proteins
5) cAMP is degraded by phosphodiesterase

What enzyme synthesizes cyclic AMP? What molecule activates this enzyme?

adenlyl cyclase. An activated alpha subunit from a Gs protein.

Where is adenlyl cyclase found?

the plasma membrane

What enzyme breaks down cAMP? Where is this enzyme found?

phosphodiesterase. Cytosol.

What is cyclic AMP synthesized FROM?

ATP

What is cyclic AMP broken down into?

AMP

How does cAMP act on protein kinase A?

cAMP binds to regulatory subunits of protein kinase A, causing the catalytic subunit to detach from the regulatory subunit and freely phosphorylate target proteins in the cell.

How does G protein signaling disruption cause cholera?

Cholera prevents G protein from hydrolyzing GTP, and the G protein never shuts off.

How does G protein signaling disruption cause whooping cough?

Pertussis toxin acts on Gi so it can no longer inhibit adenlyl cyclase, causing fluid to accumulate in the lungs.

How does G protein signaling occur with acetylcholine?

Acetylcholine binds to a muscarinic Ach receptor, activating a G protein. The beta gamma subunit of the G protein targets a sodium-potassium channel, causing potassium to enter the membrane.

What are the secondary messengers used by G proteins?

inositol trisphosphate (IP3), diacylglycerol (DAG), cAMP