biochem exam 3 study guide

What are some key differences between carriers and channels in terms of rates of transport, saturability, and stereospecificity?

transporters (carriers) are stereospecific, saturable (michaelis-menten kinetics), have transport rates much below limit of unhindered diffusion

channels have greater rates of transport, approach limit of unhindered diffusion, ion channels selective for specific ion, no stereospecificity , usually not saturable

Can carriers or channels be commonly described by Michaelis-Menten kinetics?

carriers (transporters)

What is uniport?

transporters carrying one substrate only

What is symport?

transpoters carrying two substrates in same direction

what is antiport?

transporters carrying two substrates in opposite directions

what is cotransport?

two substances are simultaneously transported across a membrane by one protein, or protein complex which does not have APTase activity

Two components contribute to the free energy stored in an ion gradient. What are they?

Chemical component of gradient
Electrical component of gradient

How are electrochemical gradients across membranes established?

When ions move across membranes and change the charges of the membrane sides

What is the difference between primary active transporters and secondary active transporters?

primary active transporters consume energy rich substrate to transport another substrate uphill a concent gradient

secondary active transporters transport one substrate down its concent gradident to transport another substrate uphill

four types of transport ATPases

P-type
V-type
F-type
ABC-type

P-type ATPases

form phosphorylated at Asp intermediate to facilitate conformational change necessary for transport function
3Na+ for 2 K+

V-type ATPase

vesicular type
acidify intracellular vesicles by transporting H+ into vesicles
can be used to store high concent of NT in synaptic or neuroendocrine vesicles

F-type ATPase

transports acting in reverse, synthesizing ATP
proton electrochemical gradient used + consumed to synthesize ATP

ABC-type ATPases

ATP binding cassette
transport many often hydrophobic molec, including lipids

Which group does the Cystic Fibrosis Transmembrane Conductance Regulator belong to?

ABC-type
chloride channel activated by ATP

Which groups does the multidrug resistance (MDR) transporter belong to?

prototype of ABC-type
pumps peptides, hydrophobic molecules,+ drugs out of cells

What are aquaporins? What are they specific for? What do they not allow to pass?

water channels that allow water but not protons to cross membranes

Name two different ways how ion channels can be opened (How can channels be classified according to their gating mechanism)?

Voltage gated - by a change in transmembrane voltage
Ligand gated - by binding of a specific chemical / ligand

How is it possible that the larger potassium is able to pass through the open potassium channel while the smaller sodium ion is not?

geometry of selectivity filter not ideal for smaller Na+ ion
Na can't compete with K+ for entry into filter

Explain the hydration status of the K+ ion before after entering the potassium channel. Describe the non-covalent interactions that occur during the passage of the K+ ion though the potassium channel and use that to explain the energetic reason for the potassium channel's ion selectivity.

K+ requied to maintain selectivity filter
two K+ ions can be accommodated in filter with one space inbetween
when 2nd K+ ion enters, electrostatic repulsion helps push first K+ out of filter, contributes to high conductance of K+ channel

Name three states of the acetylcholine receptor

closed -> open -> inactive

What opens the acetylcholine channel? Which ions pass through the open acetylcholine receptor? Describe the conformational change that allows channel opening.

Ca2+ depolarize the membrane
Na, K, Ca pass through and enter the muscle fiber
AcChR is a pentamer (nicotinic receptor in skeletal muscle) and binds two acetylcholine molecules, which opens the channel

How are lipids arranged in biological membranes (what structure do they form?)

lipids from a bilayer

What is the length of cholesterol compared to the width of a phospholipid bilayer?

cholesterol spans only one leaflet of the bilayer
found equally on both sides

How are membrane proteins classified according to their type of anchoring to the lipid bilayer? What are type I, II, III, IV, V, VI integral membrane proteins?

I - amino terminus outside
II - carboxyl terminus outside
III - span the membrane several times
IV - several polypeptide chains form a channel
V - lipid anchored
VI - lipid anchored and traverse membrane

How do lipids and proteins move in biological membranes?

move laterally
limited by proteins being anchored to cytoskeleton

In which ways are membranes asymmetric?

Proteins are glycosylated at the extracellular side, not intracellularly
Predominantly outside - sphingomyelin and phosphatidylcholine
Predominantly inside - phosphatidyl ethanolamine and phosphatidyl serine

What is needed to separate different types of membrane proteins from the lipid bilayer?

Flippase - outer leaflet to cytosolic leaflet
Floppase - cytosolic leaflet to outer leaflet
Scramblase - both directions

What is the hydropathy index? Which portions of a transmembrane protein can be predicted by this index?

Number representing the hydrophobic of hydrophilic properties of its sidechain
o The larger the number is, the more hydrophobic the amino acid
Alpha-helical transmembrane regions can be predicted by plotting the hydrophobicity of amino acids

What are two common secondary structure elements found in the transmembrane domains of integral membrane proteins?

alpha helical
beta conformation

Which lipid anchors are used to attach a lipid-anchored protein to the inner leaflet, and which to the outer leaflet of the membrane?

inner:
palmitoyl, myristoyl, farnesyl
outer:
glycosyl phosphadidyl inositol group (GPI)

Which lipids are commonly found in lipid rafts (or simply "rafts")?

sphingolipid rich microdomains
enriched in GPI anchored proteins
important role in signal transduction

What are caveolae?

inwardly curved rafts
curvature induced by caveolin, lipid anchored protein
play role in membrane traffic + signaling

In which types of cellular processes do rafts and caveolae play an important role?

signal transduction
membrane signaling + traffic

What is the difference in membrane organization of gram-positive and gram-negative bacteria?

gram pos have one membrane
gram neg have two membranes
both prokaryotic membranes

Which organelles in eukaryotic cells have two membranes (bilayers)?

mitochondria

List the most important second messengers

Ca2+
cAMP
diacylglycerol (DAG)
inositol triphosphate (IP3)

name the most important classes of receptors.

GPCR
receptor tyrisone kinases
receptor guanyl cyclases
chemically gated ion channels
nuclear steroid hormone receptors
adhesion receptors (integrins)

What are common structural features of 7TM receptors? List some of the well-known and important functions of 7TM receptors. Give an estimate how many 7TM receptors exist.

7 transmembrane helicases also called 7TM and serpentine receptors
GPCR
Several hundred different receptors are in this class, dealing with about 800 genes
Mediates signals by photons, odorants, tastants, hormones, and NT
Overall structure is similar to rhodopsin (detects photons, initiates signaling)

Which class of proteins play a key role in signal transduction of 7TM receptors (such as the b-adrenergic receptor)?

Mediates signals by photons, odorants, tastants, hormones, and NT (epinephrine, etc.)

respond to epinephrine to initiate fight or flight

Subunits of GPCR

alpha, beta, gamma

What are the most important families of G-proteins? What are their effects (they are coupled to which downstream signaling events?)

Gs: stimulate adenylate cyclase
Gi: inhibits adenylate cyclase
Gt: stimulates cGMP phosphodiesterase
Gq: increases IP3 and intracell Ca2+

How are G-proteins activated?

ligand binds to receptor
receptor is activated and activates G-protein
activate adenylate cyclase
concentration of cAMP increase
activates effectors

How are G-proteins inactivated?

ligand is dissociated
cAMP hydrolyzed by cyclic nucleotide phosphodiesterase
Ga inactivated by hydrolysis of GTP to GDP

How is the activation of 7TM receptors terminated (use the b-adrenergic receptor as an examples)?

cAMP eliminated
Gs turns itself off when GTP hydrolyzed to GDP
epinephrine signal persists to protein kinase + arrestin 2 desensitize receptor triggered by By subunits

What are the products of PLC action and what intracellular effects do these products have?

7TM receptors can be coupled to phospholipase C (PLC) via Gaq
generates IP3 and DAG from PIP2
PLC translocated from cytosol to membrane by binding to DAG

What type of receptor is the insulin receptor?

tyrosine kinase receptor

What is the name of the GTP-binding protein that is activated by the insulin receptor?

GTP binds to Ras activating it

Name three proteins that link the phosphorylated insulin receptor to the activation of this GTP binding protein.

Raf-1, MEK, ERK

Which family of intracellular signaling molecules is activated downstream of the activation of the GTP binding protein?

map kinase (MAPK), map kinase kinase (MAPKK) map kinase kinase kinase (MAPKKK)

What are the constituents (chemical components) of nucleic acids and how are they joined together? (What type of bond connects the sugars with each other and which type of bond connects the bases to the sugars?)

sugar, phosphate, base

Phosphate backbone - phosphodiester covalent bonds
Base to base - H-bond
Sugar to base - N-glycosidic bond (N1 on pyrimidine and N9 on purine)

Which of the bases are purines, which are pyrimidines? Which have a two membered ring structure, which have only one ring?

purine: adenine + guanine, 2 rings
pyrimidines: cytosine + uracil or thymine, 1 ring

What is the difference between a nucleoside and a nucleotide?

nucleoside: sugar and base
nucleotide: sugar, base, and phosphate

In which direction are nucleic acid sequences written and read?

5' to 3'

What functional groups are at the ends of nucleic acids?

5' has phosphate group
3' has hydroxyl

Why is one end called the 5' end and the other the 3' end?

refer to the number of carbon atom in a deoxyribose sugar molecule to which a phosphate group bonds

What are the chemical differences between the constituents of DNA and RNA? Name the bases that are used in DNA and RNA.

DNA: A, G, C, T, One hydroxyl group in the sugar, More stable

RNA: A, G, C, U, Two hydroxyl groups in the sugar, Less stable

Why is DNA more stable in alkaline solution than RNA?

Because DNA only has one hydroxyl group to react as opposed to RNA, which has two reacting hydroxyl groups

What are the Chargaff rules saying? Which bases form Watson-Crick base pairs?

chargaff rules: amount of A = amount of T
and amount of C = amount of G

watason-crick: A pairs with T and C pairs with G

What are the key features of the Watson-Crick DNA helix (directionality of both strands, geometry and orientation of bases and phosphates)?

DNA forms a double helix with two strands wrapped around each other
strands run antiparallel (opp directions)
phosphates point out + bases point in
bases on opp strands H bond with each other
bases are perpendicular to axis + mostly planar
10 bases per turn
DNA helix has major and minor groove

What forces contribute to DNA stability?

H bonds align bases
base stacking
hydrophobic interaction with negative phosphates on the outside and nonpolar bases on the inside

Why are there major and minor grooves? Why is their existence important?

the N-glycosidic bond between the bases and sugar don't point in exact opp directions
grooves provide access to bases inside helix and allow interact w/ proteins that bind/recognize DNA
major provides more access than minor

What are alternative conformations of double-stranded DNA? (A, B, and Z forms) What do these conformations have in common?

B- form predominant (watson + crick)
A-form: double stranded regions of RNA + some DNA-RNA hybrids
Z-form: rare

Which helix conformation is right handed, which is/are left handed?

A and B form are right handed
Z form is left handed

Which is the most common helix form?

B form

What is DNA melting and annealing, how it is monitored?

Melted - helix strands can be separated by heat (denatured)
Annealing - upon cooling, the strands come back together
Monitored by measuring the absorbance at 260 mn

How does the melting temperature depend on nucleotide (base) composition?

GC pairs are harder to melt than AT pairs

What is DNA hybridization? How does sequence (base content) influence the propensity of DNA hybridization?

similar sequences between two sample duplexes will form hybrids after being completely denatured by melting

Increases with sequence similarity between DNA1 and DNA2

What is a palindromic sequence?

DNA sequence that is identical when read backwards
same sequence in 5' to 3' and 3' to 5'

What are the requirements for the formation of a hairpin and a cruciform?

hairpins form when palindromic sequences are single stranded

cruciforms form when palindromic sequences are double stranded

List some common features of RNA structure (predominance of double- versus single-strandedness, predominance of A,B, and Z form). What type of helix is found

important funct: mRNA, tRNA, ribsomal RNA
primary mRNA transcript of DNA is single stranded and right-handed helical
can form double helix with RNA or DNA
base pairs in DNA-RNA helices: GC and AU
self complementary regions are in A form
unpaired nucleotides can from bulges or internal loops

What type of helix predominates in DNA-RNA duplexes. Which base pairs form?

double helix, single strand
GC and AU

In which direction is DNA (and RNA) synthesized in nature?

5' to 3' direction

What are the templates and the products of transcription and of translation?

transcription: DNA -> RNA, Protein synthesis does not directly occur from DNA. RNA is used as an intermediate (mRNA) produced by transcription

translation: RNA -> protein, tRNA translates the nucleic acid sequences into amino acids

templates are DNA and RNA

Does transcription or translation come first?

transcription

Consider mRNA, the coding strand and the template strand. Which is complementary to which?

mRNA template for protein synthesis
complementary to DNA sequence
mRNA and coding strand are the identical (except switch the T's for U's)
the template strand is the opposite to them both

Which nucleotides in DNA do you need to replace to get the mRNA sequence?

replace T in DNA for U in RNA

What is the name of characteristic sequence elements that indicate the start of transcription?

promoter sites
directs RNA polymerase
come before start site

Name five key characteristics of the genetic code.

1. 3 nucleotides (codon) encode one amino acid. tRNA are adaptors that translate the nucleotide sequence into amino acid sequence
2. codons are read sequentially without interruption (no overlap)
3. genetic code is degenerate
4. genetic code contains start + stop signals for protein synthesis (start: AUG - methionine stop: UAA, UAG,UGA)
5. genetic code is nearly universal

What is usually the first codon at the start of translation? What does that codon encode in prokaryotes and eukaryotes?

AUG encodes for methionine

What is monocistronic and polycistronic mRNA? How does the beginning and end of mRNA differ in prokaryotes and eukaryotes?

monocistronic: in eukaryotes, mRNA for each protein is modified after transcription to add cap + poly A tail, ribosomes bind at cap + scan mRNA until find first AUG

polycistronic: in prokaryotes, more than one protein are encoded by single mRNA (several genes on each mRNA)

Which types of molecules "encapsulate" the genetic code?

tRNA encapsulates the genetic code

What is an "anticodon"?

tRNA contains template recognition site and carries one specific amino acid

What is an intron?

non-coding intervening sequences

What is meant by the term "primary transcript"?

single-stranded RNA product to yield various mature RNA products such as mRNA, tRNA, and rRNA

How does primary transcript differ from the final (mature) form of mRNA?

The piece of DNA that is required for the synthesis of mRNA is longer and the additional pieces of DNA are required to bind the RNA polymerase enzyme and to direct it to the start

How do prokaryotes and eukaryotes differ regarding introns?

Prokaryotes - operator sequences downstream of promoters
Eukaryotes - operator sequences upstream of promoters to help regulate transcription

What is recombinant DNA technology?

series of techniques whereby segments of DNA are detected, rearranged, added, deleted, or introduced into genomes of other cells

Name four important products of recombinant DNA technology that are in therapeutic use.

human insulin, therapeutic growth factors, therapeutic monoclonal antibodies, blood clotting factors, therapeutic interferons, and many vaccines are recombinant DNA products

List six key steps that need to be taken to clone a gene

Suitable vector carrying the piece of DNA to be cloned
Cutting DNA
Joining two pieces of DNA covalently
Introducing recombinant DNA into a host cell
Selectively propagating transformed cells
Identifying host cells that contain recombinant DNA

What is the meaning of the term vector?

DNA molecule used as a vehicle to artificially carry foreign genetic material into another cell, where it can be replicated

What is the meaning of the term host?

provides enzymatic machinery for DNA replication

What is the meaning of the term transformation?

introducing recombinant DNA into host cell

What is the meaning of the term selection?

selectively propagating (cloning) transformed cells

What are restriction enzymes doing that makes them so useful for recombinant DNA technology?

generate cohesive ends or sticky ends that complementary + easily hybridized after cleavage
some can generate blunt ends
sticky and blunt ends can be re-joined by ligases

What is the meaning of the term palindromic sequence?

same when read left to right and right to left

What is the meaning of the term sticky ends?

complementary and easily hybridized after cleavage

What is the meaning of the term blunt ends?

no unpaired bases / overhangs in the end so both strands are the same length

What do most restriction sites have in common (sequence characteristics)?

Sticky and blunt ends can be rejoined (recombined) by ligases if they were cleaved with the same restriction enzyme

What is a plasmid and why is it useful for cloning?

plasmids occur naturally in bacteria, can be used as cloning vectors, are exchanged between bacteria (conjugation) and are extrachromosomal circular DNA, self replicating, carry genes for resistance for antibiotic drugs

bacteria from which plasmids are isolated grow quickly and make more of the plasmids as they grow