Mmbio Final New Version

Front 1

In eukaryotes, a protective cap made from what is added to which end of the mRNA. WHat other modifications are made at this end?

Back 1

guanosine is added to the 5' end. It is a methylated guanosine (7-methyl guanine)

Front 2

What is added to the 3' end of mRNA?

Back 2

A poly A tail

Front 3

what is unique about the 5' cap? Why is this important

Back 3

The is a 5' to 5' triphosphate linkage (with GTP). it prevents recognition by exoribonucleases (which can digest RNA from either end)

Front 4

Why do eukaryotes add 3' tails and 5' caps

Back 4

to help prevent the degradation of mRNA and helps regulate the mRNA. They also directly participate in translation because they interact with the ribosome.

Front 5

Following the cap, what is methylated?

Back 5

the guanine and the 2' OH groups of the fist and second nucleotides.

Front 6

What is the name of the capping enzyme? Only RNAs made by what are capped and why?

Back 6

guanylytransferase, only RNAs made by pol II because they are associated with the CTD of Pol II.

Front 7

What is the purpose of the PolyA tail?

Back 7

This tail isbound by poly A binding protein to protect the mRNA from 3’ -5’ exonucleasedegradation. This polyA tail-protein complex can then associate with the 5’ endof the mRNA and cap-binding protein to increase the efficiency of translationof the mRNA. It may also influence subsequent stages of mRNA maturation andfunction.

Front 8

What enzyme is responsible for addition of the polyA tail?

Back 8

Transcription by RNA polymerase II proceeds until itcopies the poly(A) addition site sequence, to which polyadenylation factorsbind and cleave the mRNA. Poly A polymerase adds a poly A tail (or polyadenylation).

Front 9

Swi/Snf

Back 9

chromatin remodeling complexes that are able to reposition, eject and unwrapnucleosomes, and exchange or eject histone dimers from the chromatin.

Front 10

Are remodeling complexes are ATP-dependent?

Back 10

Yes, they are

Front 11

Name the three yeast proteins required for thecontrol of gene expression by galactose

Back 11

Gal4p, Gal80p, and Gal3p

Front 12

What are the functions of Gal4p, Gal80p, and Gal3p in yeasts?

Back 12

Gal4p is the transcriptional activator that binds to the GAL gene upstream activator sites, this promotes the transcription of the gene.

Gal80p is an inhibitor that binds and blocks the trans-activating region of the Gal4p when galatose is low.

Gal3p binds to Gal80p when galactose (the effector) is present. This causes a conformational change that allows the Gal4p to act as an activator again because it deactivates Gal80p

Front 13

What are the six transcriptional regulation mechanisms or elements that are unique to eukaryotes (not in bacteria)

Back 13

Insulators that restrict activation to the target promoter and prevent activation of adjacent genes

Enhancesomes

Gene silencing

Imprinting

Dosage compensation (ie barr bodies)

hormone signals sent by other cells

Front 14

what controls the transport of mRNA from the nucleus to the cytoplasm?

What is the most important protein in the EJC?

Back 14

Ran proteins bind to the mRNA and associate with exportin; the complex is then exported through the nuclear pore. only processed mRNA can leave, the cell can tell the difference because processed mRNA has EJC put on them during splicing.

REF1 is part of the EJC

Front 15

Splicing generates a specific mRNA protein that targets mRNA for nuclear export

Back 15

EJC

Front 16

When I is in the first position of the anticodon of tRNA it binds to

Back 16

It can bind to A C and U

Front 17

How do eukaryotic transcription factors exert their effects on transcription when their binding sites can be thousandsof base pairs away from the promoter?

Back 17

The DNA between the bound site and the polymerase-promoter complex forms a loop, which brings them into proximity.

Front 18

What is the role of the branch sequence in splicing of nuclear introns? What base in the branch sequence is essential for splicing, and what makes this particular base so reactive?

Back 18

The branch sequence is near the end of each intron, and pairs with the U2 snRNP resulting in a bulge with an A that doesn’t pair with the U2 snRNA. The 2’ OH on this A is then exposed and is very reactive, and carries out the attack onthe exon-intron boundary as the first reaction in the intron splicing pathway.

Front 19

what are the splice sites for nuclear splicing? (with the splicisome)

Back 19

GU and AG.

Front 20

What is the branch point and its significance?

Back 20

An A that does not base pair, so it's 2' oh group forms a unique 5' to 2' linkage

Front 21

What is the basic process of spliceosome assembly

Back 21

1. U1 binds to the 5' splice site (GU) and U2 binds to the branch point (A).

2. The U4-U5-U6 trimeric snRNP displaces U1 at the 5' splice site, the U4 dissociates

3. U6 and U2 catalyze attack of the branch point on the 5' splice site (AG) --this cuts one exon away from the intron lariat loop

4. the 5' splice site attacks the 3'splice site, which separates the other exon

Front 22

RememberU1—binds to

U2—binds to

U5—binds to

Back 22

Remember U1—binds to AG site, U2—binds to U1 and the branch point, U5—binds to both sides of the intron and then seals the exons together

Front 23

What is required for recognition of splice junctions and correct excision of introns?

Back 23

Small ribonucleoproteins that each recognize a part of the intron sequence and structure.

Front 24

Group I introns are found mostly in

how is unique from nuclear splicing ?

Back 24

single-cell eukaryotes and can be self-spliced without any protein or additional RNA factors

The folding of the RNA molecule brings the exons together and exposes the intron to self-splicing (ribozyme) activity.

Front 25

The mechanism of self-splicingof group I introns

Back 25

1. the 3' OH of guanosine acts as a nucleophile, attacking the phosphate at the 5' splice site.

2. the 3' OH group of the 5' exon becomes the nucleophile, completing the reaction

Front 26

What is required to initiate two transesterification steps in the mechanism of self splicing? Group 1

Back 26

exogenous guanosine, it acts as the nucleophile, making the first cut

Front 27

Group II introns are commonly found in

Back 27

chloroplasts and in mitochondria of plants and fungi.

Front 28

Which type of splicing does not require a lariat? What is needed for this to be possible

Back 28

Group 1

Front 29

Mechanism for group II splicing

Back 29

No snRNPs arerequired,but otherwise the process is very similar to nuclear intron splicing. The folding of the RNA molecules (secondary structure) provides the same function that is normally met by the spliceosome snRNPs

Front 30

Many human mRNAs are edited by

Back 30

adenosine deaminase acting on RNA (ADAR).

Front 31

C to U editing of the mRNA for ApoB protein results in

Where is C to U editing found?

Back 31

different length proteins in the liver and intestine. The shorter protein aids in absorption of lipids in the intestine.

Its found in many transcripts in plant mitochondria and chloroplasts.

Front 32

In animal cells, the most common form of RNA editing occurs by

Back 32

Deamination of a C to create a U or of A to create an I (inosine),without cutting the RNA chain.

Front 33

RNA export and import from/to the nucleus through the nuclear pores is controlled by

Back 33

exportin and importin proteins and GTP

Front 34

What is the start codon?

Back 34

AUG

Front 35

What are the three stop codons?

Back 35

UAA, UAG, and UGA

Front 36

What is the wobble hypothesis?

Back 36

It describes how a single tRNA can bind to more than one codon

1. the first base pair of the anticodon (third of codon) allows one tRNA to recognize more than one different codon (I, G, and U)

2. first two base pairs on codon do not wobble

3. a minimum of 32 mRNAs are require

Front 37

Inosine is a modification of what?

Back 37

adenine

Front 38

How many possible reading frames for amino acids are there in any mRNA?

Back 38

There are three. **The stop codon must be in the same reading frame as the start codon for the open frame

Front 39

Suppression of a nonsense mutation.In the wild-type, CAG encodes glutamine. A nonsense mutation changing this to UAG would create a premature stop (nonsense) codon. A tRNA suppressor has a mutant anticodon that pairs with the UAG nonsense (stop) codon resulting in a full-length protein with tyrosine instead of glutamine.

Back 39

just memorize this..

Front 40

Proteins make up what % of the dry weight of a human body

Back 40

44%

Front 41

Where are ribosomes located?

Back 41

In the cytoplasm, or attached to the endoplasmic reticulum

Front 42

what are essential for ribosome structure and function, and are highly conserved in different organisms.

Back 42

rRNAs

Front 43

Three steps of translation

Back 43

initiation, elongation, termination

Front 44

what are polysomes?

Back 44

multiple ribosomes associated with a single mRNA

Front 45

On a sucrose density gradient , what lies on the far right of the graph (heavy side)

Back 45

Polysomes comprising two or more ribosomes associated with mRNA transcripts migrate in the heavier fractions of the gradient.

Front 46

In what organism are translation and transcription coupled? why?

Back 46

In bacteria, transcription and translation can be coupled because they both occur in the cytoplasm

Front 47

Ribosomes have three sites:

Back 47

A (aminoacyl), P (peptidyl), and E (exit),

Front 48

Alignment of tRNAs on the mRNA and the peptidyltransferase site. The anti-codon-codon binding causes

Back 48

kinks in the mRNA

Front 49

Where does the polypeptide chain exit the ribosome?

Back 49

the exit tunnel, which is in the large subunit (50s)

Front 50

What is used when attaching an amino acid to a specific tRNA

Back 50

ATP hydrolysis, a Mg2+ dependent enzyme: aminoacyl-tRNA sythetase

Front 51

What is charged tRNA?

Back 51

tRNA that has an amino acid attached to it, the amino acid will be attached at the 3' terminal adenosine

Front 52

How are tRNAs charged?

Back 52

figure 18-11

Front 53

What are the five arms of tRNA and what do each of them do?

Back 53

D arm-stemp-loop structure, has dihydrouridine groups, the recognition site for aminoacyl-tRNA synthetase

Extra arm (variable arm)-is the size difference between tRNA molecules, has between 4 and 21 bases.

Anticodon arm-stem-loop structure, base pairs with the mRNA

Acceptor/Amino acid arm-the site of amino acid attachment to tRNA (at 3' end), it has a double stranded region, and a single-stranded CAA sequence

TC arm- stem loop structure, the ribosome binding site

Front 54

Proofreading by an aminoacyl-tRNA synthetase.

Back 54

An amino acid that is too large will not fit in the acylation site (a), and thus is blocked. If a smaller amino acid enters the site, it will sometimes charge the tRNA, but the smaller form fits into the synthetase’s proofreading site, where it is hydrolyzed to release it from the tRNA.

Front 55

How does the mRNA bind to the ribosome in bacteria?

Back 55

It binds to the 16s sequence of the rRNA in the small ribosome subunit 30S

Front 56

Describe translation initiation in bacteria

Back 56

IF-1 binds to the A site, IF-3 binds to the E siteto prevent premature binding of the elongation aminoacyl-tRNAs and premature binding of the 50S subunit. IF-2 hydrolyzes charged fMEt-tRNA to get it to the initiation site, which makes the ribosome assemble

Front 57

IN ribosome terms, 30S + 50S =

and 40S + 60S=

Back 57

70S, 80S

Front 58

What happens to the tRNA for methionine in bacteria that doesn't happen in eukaryotes?

Back 58

In bacteria, there is a special initiator tRNA formethionine, and the methionine is modified by theaddition of a formyl group bymethionyl-tRNA formyltransferase. It has a specific tRNA for it, other methionines found within the gene have a different tRNA attached.

Front 59

What is the Kozak sequence?

Back 59

a sequence found in eukaryotes that is similar to the shine delgarno sequence, it is not necessary, but it does enhance translation efficiency (not involved in base pairing like the shine delgarno sequence

Front 60

describe translation initiation in eukaryotes

Back 60

EIF4F recognizes the 5' cap of processed mRNA, the binding recruit the small ribosome subunit and initiator methionine tRNA to assemble on the mRNA by looping to identify the first AUG codon

Front 61

how and why does circularization of eukaryotic mRNAs happen?

Back 61

the 5' cap and the polyA binding proteins interact (the eIF4F is still attached). This increases effciency of translation

Front 62

Where do peptide bonds form? In which ribosomal site do they grow?

Back 62

they form between amino acids in the P and A sites, they grow in the P site

Front 63

Describe each elongation factor (the T ones)

Back 63

EF-Tu binds GTP and brings each new tRNA into the ribosome A site during protein synthesis, when the GTP gets hydrolyzed to GDP, it EF-Tu leaves

EF-Ts recycles the EF-Tu after GTP hydroysis so the EF-TU can bind another tRNA (takes off GDP and adds GTP)

Front 64

EF-G

Back 64

EF-G is responsible for translocation of the ribosome along the mRNA, codon by codon, and also hydrolyzes GTP to provide energy for translocation (basically pushes the protein out of the A site and leaves it empty)

Front 65

How does termination of translation happen in bacteria? Talk about release factors

Back 65

When the ribosome comes to a stop codon, RF-1 or RF-2 binds to the A site, which induces the release of the polypeptide. RF-3 then comes and used GTP to release RF-1 or RF-2

Front 66

How are ribosomes recycled in bacteria?

Back 66

Ribosomes are recycled (left). In bacteria, ribosome recyclingfactor (RRF) andEF-G separate the ribosome from themRNA andtRNAs, andIF-3 associates with the 30S subunitagain. RRF has a similar shape to tRNA,whichallowsit to bind to the tRNAsites in the ribosome

Front 67

What is the major source of energy for ribosome translocation and termination of translation?

Back 67

GTP

Front 68

How can amino acids be modified? Why would they be modified?

Back 68

phosphorylation, carboxylation, or methylation. They are modified after translation to alter the function of the protein

Front 69

What happens if an mRNA is partially degraded, so it is shorter than it should be? What will happen to the ribosome when it gets to the end?

Back 69

The ribosome stalls, so tmRNA has to come to same the day, it does so by mimicking both mRNA and tRNA. It marks the truncated polypeptide for degradation

Front 70

Describe the process of tmRNA

Back 70

Front 71

look at slide 37 chapter 18

Front 72

For a newly synthesized protein, what are its properties/sequence that determines its final location in the cell? What is the default location of a new protein if it lacks any of these other determinants?

Back 72

Proteins often have an N-terminal localization or targeting sequence (TS), that is recognized by other proteins in the cell to facilitate movement of the protein to its proper location, such as the endoplasmic reticulum, mitochondria, etc. N-terminal TS are usually cleaved off once the protein has reached its proper destination. An exceptionis nuclear proteins, the nuclear localization signal (NLS) is usually internal and not cleaved.

If a protein does not have a TS or other localization signal, it will remain in the cell cytoplasm where it was synthesized (Many proteins do not need processing and remain in the cytoplasm).

Front 73

Describe the basic process of trafficking proteins from the cytosol into the endoplasmic reticulum

Back 73

polypeptide has a signal sequence, SRP attaches to the signal sequence which causes it to bind to the SRP, which is located by the peptide translocation complex (translocon) on the membrane of the ER lumen.

Front 74

if you have time, look up targeting of nuclear proteins

Back 74

slide 41 ch 18

Front 75

how do the proteins of the transcription machinery access DNA that is tightly associated in chromatin?

Back 75

remodeling complexes (swi/snf), master transcription factors may be bound to some promoter regions to block nucleosome formation at the promoter sequence so it can interact with other transcription factors. Remodeling complexes are ATP-dependent

Front 76

Insulators

Back 76

transcriptional gene regulator only found in eukaryotes, it makes sure that enhancers only bind to their matching promoter (no mixed signals)

Front 77

Imprinting

Back 77

transcriptional gene regulator only found in eukaryotes. Completely shutting down the expression of the allele derived from one parent because the insulator binds to it. Methylated insultor sites are on the exoressed allele

Front 78

Dosage compensation

Back 78

transcription mechanism in eukaryotes. Inactivates an X chromosomes (bar body in mammals covered by XIST), double expresses the male X chromosome, on expresses each X chromosome by 1/2

Front 79

Hormone signals

Back 79

transcription regulation in eukaryotes. Hormones diffuse across membranes to activate transcription factors

Front 80

enhancesomes

Back 80

transcription regulation only found in eukaryotes. big protein complex that has favorable interactions with other proteins and helps activate genes

Front 81

How does termination of transcription of mRNA molecules occur (i.e. what sequences and/or proteins are involved)? What is found at the 3’ end of nearly all eukaryotic mRNA molecules? What are two functions of this modification?

Back 81

Transcription by RNA polymerase II proceeds until it copies the poly(A) addition site sequence, to which polyadenylation factors bind and recruit cleavage factors that cleave the mRNA (RPase II proceeds a little further and then dissociates from the DNA). Poly Apolymerase adds a poly A tail (or polyadenylation). This tail is bound by poly A binding protein to protect the mRNA from 3’ -5’ exonuclease degradation. This polyA tail-protein complex can then associate with the 5’ end of the mRNA and cap-binding protein to increase the efficiency of translation of the mRNA. It may also influence subsequent stages of mRNA maturation andfunction.

Front 82

Alternate splicing

Back 82

Alternate splicing occurs when one combinationof exons in a primary (or pre-) mRNA is incorporated into one mature mRNA whileother combinations of exons are incorporated into other mRNAs for the samegene. Alternate splicing occurs when one or more splicing sequences in atranscript are passed over by the spliceosome, leading to the inclusion of anintron as part of an exon in some transcripts, or to deletion of an exon aspart of an intron in other mRNAs. This can lead to multiple mRNAs from the samegene

Front 83

How prevalent is alternate splicing in the human transcriptome?

Back 83

It is estimated that more than 90% of human pre-mRNAs can undergo alternate splicing, contributing to thewide number of different proteins encoded in the human genome

Front 84

What type of bond is involved in the attachment of amino acids to tRNA molecules? What enzymes carries out this reaction?

Back 84

The aminoacyl group of the amino acid is attached tothe 3’ terminal adenosine of the tRNA (at the end of the CCA sequence) by anester bond between the α carboxyl on the aminoacylgroup and the 2’ or 3’ hydroxyl group of the adenosine.

The enzyme is called aminoacyl-tRNA synthetase

Front 85

proofreading of the aminoacyl-tRNA synthetase

Back 85

It requires ATP for activity and has proofreadingactivity to ensure the correct amino acid is attached. Small differences in theenzyme and the tRNA determine specificity.

Front 86

why do some ribosomes associate with the ER to form the rough ER while others are free in the cytoplasm?

Back 86

It all depends on the protein they are making--if it has a N-terminal signal sequence (20-30bp, positively charged) the ribosome will be anchored to the ER. If proteins are meant to stay in the nucleus, mitochondria, or other organelles are translated in the free ribosomes.

Front 87

What challenges are associated with thetranslation of incomplete mRNAs or mRNAs cleaved by a nuclease, both of whichlack a termination codon? How do the cells deal with this potential problem?

Back 87

The ribosome is stalled, at the incomplete 3' end of the mRNA. BACTERIA use tmRNA to fix the problem. The tmRNA binds to the A site and it continues to translate a short peptide, it reaches a stop codon, is released, then the proteins is degraded. RIBOSOME RESCUED

Front 88

How is tmRNA like tRNA?

Back 88

It is charged with alanine, alanyl-tRNA sythetase charges it.

Front 89

What is the name of the short peptide made when using tmRNA?

Back 89

proteolysis tag, which leads the mRNA to be degraded.

Front 90

Intron splices sites in primary transcripts(pre-mRNA) are recognized in a eukaryotic cell nucleus by the spliceosome via:

Back 90

base pairing with snRNAs that are part of the snRNPs

Front 91

How do eukaryotic transcription factors exert their effects on transcrition when binding sites can be thousands of base pairs away from the promoter?

Back 91

The DNA between the bound site and the polymerase-promoter complex forms a loop, which bring them into proximity

Front 92

How do eukaryotic ribosomes recognize mRNA and determine where to initiate translation?

Back 92

The 5' cap is recognized by the eIF-4F, which also binds to other sequences near the 5' end of the mRNA. This binding recruits the small ribosome subunit and initiation factors, and scans the mRNA by looping to identify the first AUG codon in the mRNA, which will be the start codon.

Front 93

The growing peptide chain is found in the

Back 93

p-peptidyl site

Front 94

In what part of the eukaryotic cell does intron splicing 5' capping and 3' polyA addition occur?

Back 94

the nucleus

Front 95

The structure of the U1 snRNP

Back 95

has an SM core domain near the 3' end and a U1 C protein binding site near the 5' end

Front 96

What is RNA editing

Back 96

one or more bases in the RNA are changed to generate the proper codon for translation, this occurs in single-celled eukaryotes by insertion and deletion

Front 97

amino acids bind to which end of the tRNA

Back 97

the 3' end of the amino acid/terminal arm

Front 98

Nucleosomesin heterochromatin

Back 98

have H1 bound and are tightly packed together, so the DNA is transcriptionally silent

Front 99

Nucleosomes in euchromatin

Back 99

space further apart, is accessible for for binding by transcription factors and other proteins

Front 100

Cyclic AMP-dependent protein kinase A (PKA) is repressed by a

Back 100

Regulatory subunit of the adenylate cyclase holoenzyme and becomes active only on binding of cAMP. The cAMP is produced when a signal molecule binds a trans-membrane receptor and induces it to activate adenylate cyclase. Once active, PKA catalytic subunits enter the nucleus and phosphorylates target proteins and recruits RPase.

Front 101

Using western blot, what is one fairly simple exampleof how some genes are activated at just the right time during development,and then are turned off.

Back 101

Western blot analysis shows the patternof transcription factors involved in thedifferentiation of myoblast cells tomyotubules in mouse muscle tissue.

Front 102

RNAi

Back 102

short double-strand RNA leaves the nucleus, is separated by a dicer, then carried by an argonaut and used to target specific squences of mRNA for degradation