Lecture 5

Question 1

Another mode of post-transcriptional gene regulation has been discovered that involves small double-stranded RNA

Answer 1

RNA interference

Question 2

Mammalian cells normally express short double-stranded RNAs, termed microRNA (about 500 different miRNA), that are involved in –.

Answer 2

gene silencing

Question 3

– are important during development, for example to silence particular genes and allow proper differentiation of specific cell types

Answer 3

MicroRNAs

Question 4

– can also be introduced into cells to silence a targeted gene. This allows an easy method to turn off expression of a specific gene. Such “targeted” RNAi has therapeutic potential to treat diseases such as cancer and infection by RNA viruses such as HIV

Answer 4

Exogenous double-stranded RNA

Question 5

There are four basic components of RNAi – the RNA, two processing –, and the RNA-induced Silencing Complex (RISC) that actually carries out silencing

Answer 5

RNases

Question 6

Endogenous microRNA is synthesized as part of a longer RNA by –. The microRNA sequences are homologous and base-pair, forming a “stem” in the precursor.

Answer 6

RNA pol II

Question 7

Two RNases, one in the nucleus and one in the cytoplasm, cleave the – to produce the short (21-25 nucleotide) double-stranded RNA

Answer 7

precursor

Question 8

the cleaved RNA is unwound and one strand associates with an– which is part RISC

Answer 8

Argonaut protein,

Question 9

The single-stranded microRNA targets – to mRNA that contain homologous sequences by base-pairing.

Answer 9

RISC

Question 10

Protein production from the mRNA is prevented either by –or by RISC-mediated inhibition of mRNA translation. RNAi can also inhibit transcription, but the mechanism has not been defined.

Answer 10

mRNA degradation

Question 11

The new technique CRISPR/Cas9 allows changes to be precisely made in –

Answer 11

genomic DNA.

Question 12

Using this technique, nucleotides can be inserted, deleted, or changed in the genome

Answer 12

CRISPR/Cas9

Question 13

CRISPR/Cas9 , it allows targeted mutation of specific genes or sequences, such as those that cause cancer, or – of inherited or somatic mutations that cause disease

Answer 13

repair

Question 14

CRISPR

Answer 14

clustered regularly interspaced short palindromic repeats

Question 15

CRISPR ) refers to arrays of – found in bacteria that play a role in defending bacteria against invasion by viruses

Answer 15

repeated sequences

Question 16

Within CRISPR repeats are sequences – to sequences in bacteria viral DNA.

Answer 16

homologous

Question 17

RNA transcribed from the CRISPR sequences (and processed) binds to – and targets that nuclease to invading viral DNA by base-pairing.

Answer 17

a Cas (CRISPR-associated) nuclease

Question 18

the CRISPR system has been adapted to modify mammalian genomes using a specific version of the Cas nuclease, –, and an engineered guide RNA that consists of sequences homologous to the target sequence and sequences that bind to Cas9.

Answer 18

Cas9

Question 19

Expression of the – and Cas9 in a cell directs the nuclease to sequences in the genome that are homologous with the guide RNA.

Answer 19

guide RNA

Question 20

Once the guide RNA base-pairs, the Cas9 nuclease can – This can cause a deletion, and inactivate the gene

Answer 20

cleave both strands of the DNA.

Question 21

However, if a synthetic DNA strand homologous to the targeted region is introduced, this DNA is used to – the cleaved genome DNA.

Answer 21

repair

Question 22

If the synthetic DNA contains a slightly different sequence, the new sequence is – into the genome.

Answer 22

inserted

Question 23

Eukaryotic cells are organized into membranous compartments (–) that have unique components and functions.

Answer 23

organelles

Question 24

Two important interconnected membrane systems involved in – are the secretory pathway and the endocytic pathway.

Answer 24

protein transport

Question 25

The secretory pathway consists of the -- and Golgi complex.

Answer 25

endoplasmic reticulum (ER)

Question 26

The secretory pathway is responsible for transport of --proteins to the appropriate compartment:

Answer 26

newly-synthesized

Question 27

Secretory pathway transports proteins -- (e.g. peptide hormone and growth factors such as insulin and epidermal growth factor, components of the extracellular matrix such as collagen, carrier proteins in the blood such as albumin, etc.)

Answer 27

out of the cell

Question 28

secretory pathway transports plasma membrane proteins to the -- (e.g. ion channels, hormone receptors, transporters)

Answer 28

cell surface

Question 29

T/F: secretory pathway transports proteins to lysosomes (degradative enzymes of lysosome)

Answer 29

true

Question 30

transporting proteins to secretory pathway organelles (e.g. -- and glycosidases in ER and Golgi).

Answer 30

glycosylases

Question 31

The endocytic pathway consists of --(early and late) and ends at the lysosome.

Answer 31

endosomes

Question 32

The endocytic pathway is involved in uptake of material from the -- and the extracellular fluid. It is important for uptake of nutrients such as cholesterol and iron, as well as internalization of signaling receptors.

Answer 32

plasma membrane

Question 33

This pathway is also used by certain pathogens such as viruses to enter the cell

Answer 33

endocytic pathway

Question 34

Extensive network of interconnected membrane sacs and tubule

Answer 34

endoplasmic reticulum

Question 35

Smooth ER – a major site of -- synthesis

Answer 35

fatty acid and lipid

Question 36

Rough ER – studded with ribosomes, site of -- that enter the secretory pw (also some lipid synthesis) and protein glycosylation.

Answer 36

protein synthesis

Question 37

Golgi complex – complex organelle with both -- regions and stacked membrane disk called cisternae

Answer 37

tubulated

Question 38

Organized into cis Golgi network (closest to ER), --, and trans Golgi network (closest to PM).

Answer 38

cis, medial and trans Golgi cisternae

Question 39

Golgi is a site of protein glycosylation and -- side chains. Also major site of protein sorting.

Answer 39

modification of carbohydrate

Question 40

First step in protein transport through secretory pathway: protein synthesis and -- across (or into) ER membrane.

Answer 40

translocation

Question 41

Second step in protein transport through secretory pathway: -- and modification in ER

Answer 41

Protein folding

Question 42

Third step in protein transport through secretory pathway: Transport to Golgi, lysosomes, plasma membrane by budding and --

Answer 42

fusion of small transport vesicles.

Question 43

All proteins except those made in the mitochondria are synthesized on --

Answer 43

cytoplasmic ribosomes.

Question 44

Proteins are targeted to membrane organelles by --

Answer 44

sorting signals

Question 45

Targeting relies on a short stretch of -- in the nascent polypeptide that directs it to the appropriate compartment. Referred to as a sorting signal because it is responsible for sorting to a specific compartment.

Answer 45

amino acids

Question 46

In the case of ER targeting, the sorting signal is --

Answer 46

signal sequence

Question 47

Signal sequence is normally located at the -- of the protein and consists of one or more positivelycharged amino acids followed by 6-12 hydrophobic amino acids

Answer 47

N-terminus

Question 48

Signal sequence is recognized by --

Answer 48

Signal Recognition Particle (SRP)

Question 49

SRP is an -- with one RNA and multiple polypeptides

Answer 49

ribonucleoprotein particle

Question 50

SRP binds to the large ribosomal subunit and the signal sequence as emerges from ribosome, --

Answer 50

pauses translation.

Question 51

SRP-ribosome-nascent polypeptide complex is then bound by--, an ER membrane protein.

Answer 51

SRP receptor

Question 52

Both SRP and SRP receptor are -- – GTP binding by both increases affinity of SRP for SR.

Answer 52

GTPases

Question 53

Once bound to SRP receptor, the ribosome and nascent chain are transferred to the --, a protein that forms a gated channel through the ER membrane.

Answer 53

translocon

Question 54

SRP and SRP receptor hydrolyze GTP and --

Answer 54

dissociate.

Question 55

Continued protein synthesis pushes the polypeptide in an unfolded state through the translocon channel into the lumen of the ER. The signal sequence is cleaved by a --during translocation

Answer 55

protease

Question 56

Newly synthesized membrane proteins integrate into the ER from the translocon. Same basic process but a --within the protein is transferred to the membrane from the translocon, resulting in an integral membrane protein

Answer 56

hydrophobic sequence

Question 57

N-linked glycosylation in the ER. Proteins undergo N-linked glycosylation during translocation and --, including formation of disulfide bonds, in the lumen of the ER.

Answer 57

fold

Question 58

Glycosylation involves the addition of a preformed 14 sugar oligosaccharide from a -- donor that is embedded in the ER membrane through the lipid (dolichol).

Answer 58

lipid linked

Question 59

The sugar is transferred in block as the target amino acid sequence (asparagine –X – serine/threonine) emerges from the translocon. This -- is conserved in all eukaryotic species.

Answer 59

“core” oligosaccharide

Question 60

Core is trimmed by -- in the ER.

Answer 60

glycosidases

Question 61

Folded, glycosylated proteins are transported from the ER to the Golgi by transport vesicles that bud from the ER and fuse with the --.

Answer 61

cis Golgi network

Question 62

Many other transport steps in the secretory pathway are mediated by --

Answer 62

transport vesicles

Question 63

Vesicles also mediate transport through the --.

Answer 63

endocytic pathway

Question 64

Best characterized vesicle transport pathways involve vesicle with protein coats – referred to as --

Answer 64

coated vesicles

Question 65

Coat formation: polymerization of specific proteins onto the cytoplasmic face of the organelle membrane forms a “coat” that drives -- and collects proteins (cargo) for transport to the next compartment.

Answer 65

membrane curvature

Question 66

Sorting signals (normally 4-6aa in length) on the -- of cargo bind to coat proteins, directing the cargo into the forming vesicle.

Answer 66

cytoplasmic domains

Question 67

Once the vesicle buds from the donor compartment, the coat is --

Answer 67

released (uncoating)

Question 68

(Golgi to ER, intra-Golgi)

Answer 68

COPI

Question 69

ER to Golgi

Answer 69

COPII

Question 70

Golgi to endosomes; endocytosis

Answer 70

clathrin

Question 71

Vesicles are targeted to the correct recipient organelle membrane by two classes of proteins: --

Answer 71

tethers and SNARES

Question 72

-- provide the initial link between vesicle and target

Answer 72

tethers

Question 73

SNARES are integral membrane proteins present on both the vesicle (v-SNARE) and target membrane (t-SNARE) and bind each other to drive --

Answer 73

membrane fusion

Question 74

v-SNAREs and t-SNAREs form parallel --bundles that force the vesicle and target membrane together.

Answer 74

alpha helical

Question 75

Different sets of tethers and SNAREs used at each transport step, providing -- in docking and fusion.

Answer 75

specificity

Question 76

Vesicle mediated: After proteins are transported from the ER to the Golgi, --oligosaccharides are modified by glycosidases (cleave sugars) and glycosylases (add sugars).

Answer 76

N-linked glycoprotein

Question 77

Vesicle mediated: -- is a major site of protein sorting

Answer 77

trans Golgi network (TGN)

Question 78

Secreted proteins sorted to constitutive secretory vesicles or, in specialized cells to regulated secretory vesicles or granules (e.g. insulin, glucagon, neurotransmitters, digestive enzymes). Regulated secretory vesicles only fuse to plasma membrane if cell receives the appropriate --

Answer 78

extracellular signal.

Question 79

Lysosomal enzymes sorted into --coated vesicles that fuse with endosomes.

Answer 79

clathrin

Question 80

Clathrin coats also contain “adaptor complexes” which link clathrin to membrane and bind --

Answer 80

cargo

Question 81

A -- on N-linked oligosaccharides is the sorting signal on lysosomal enzymes

Answer 81

sugar modification

Question 82

Defects in lysosomal enzyme sorting due to mutation of GlcNAC phosphotransferase cause recessive inherited lysosomal storage disease called --

Answer 82

I-cell disease.

Question 83

Without --, lysosomal enzymes are not sorted into clathrin coated vesicles at the TGN but are secreted from the cell.

Answer 83

M6P

Question 84

in I-cell disease, lysosomes do not contain --of the enzymes used for degrading macromolecules

Answer 84

normal levels