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Björn Usadel.
Worringer Weg 1
52062 Aachen
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Email: usadel /ta/ mpimp-golm.mpg.de
usadel /ta/ bio1.rwth-aachen.de

Phone: +49 241 80 26634
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MB 4 BioT Quizz

Packaging of DNA

What is so remarkable about RNA that made some people speculate about an RNA world?

 

RNA can act as an enzyme and can serve as a store of genetic information.

 

 

Why does DNA need to be packaged?

 

DNA as a molecule is very long. It needs to fit into a cell or into the nucleus

 

 

Do bacteria have histones?

 

No but they have similarly positively charged proteins, like IHF (Integration host factor)

 

 

What are the four histones making up the core particle?

 

H2a, H2b, H3, H4

 

 

Where does H1 come into play?

 

As an outer particle, additional packing

 

 

How long (in nt) is a stretch of DNA around a nucleosme?

 

~146b

 

 

How can one determine the length of DNA around a nucleosome?

 

micrococal DNAase digestion, the nucleosme protects the DNA, then run the fragments on a gel

 

 

If you digest chromosomal DNA with micrococal DNA and run them on a gel you get shortish fragments of around 170-200bp but also longer and much longer bands, where are these coming from?

 

incomplete digestion

 

 

What histone modifications do you know?

 

–Acetylation (Ac)

 

–Ubiquitination (Ub)

 

–Methylation (Me)

 

–Phosphorylation (P)

 

–Sumoylation (Su)

 

 

What does HAT stand for?

 

Histone acetylase

 

 

What does PRC2 and PRC1 do and how?

 

PRC2 (polycomb repressive complex) silences genes via histone modification, PRC1 maintains this silencing state

 

 

What is LHP1?

 

A plant PRC1 like protein

 

 

What is H3K27me3 short for and with what is this histone modification associated?

 

Histone 3 Lysine 27 triple methylated

 

It is associated with genes.

 

 

Which chemical modification of DNA do you know?

 

Metyhlation of Cytosines

 

 

Sometimes "packed" chromosomal DNA needs to be assessed, how is this problem solved?

 

By nucleosome remodelling factors. These can change the localization of the nucloesomes with respect to the DNA

 

 

What does a bromo- and what does a chromodomain do, in the context of binding to certain structures?

 

-Bromodomain (binds acetylated lysines)

 

-Chromodomain (binds methylated lysines)

 

 

Why is DNA methylation risky?

 

Methylated cytosines are less chemically stable, so more likely to undergo deamination, which changes the base to thymine (also a valid base)

 

 

What is DNA methylation used for in bacteria?

 

It distinguishes new from "old" DNA (new DNA is hemimethylated) also it distinguishes foreign from own DNA

 

 

What are restriction enzymes?

 

Enzymes recognizing specific sites in the DNA and cutting the DNA

 

 

Do all eukaryotes use the same DNA methylation sites?

 

No animals use mostly CG sites, Plants CG, CHG, CHH

 

 

What is the advantage of CG methylation sites?

 

These are symmetric, so maintaince methylation is easy

 

 

how is the X chromsome inactivated in mammals?

 

Through silencing

 

 

Explain position effect variegation

 

See the Drosophila example

 

 

How are transposons kep quiete by the host?

 

Through silencing

 

 

what happens to plants that can no longer methylate DNA?

 

Transposons get reactivated, and subsequently the plants get less and less fit

 

 

Explain paramutation using one example?

 

See the maize B-I example

 

 

What are point/region/holocentric centromers?

 

Basically this defines the centromer sites, point centromers are very small, regoin centromers hundreds of kilobases, in holocentric centromers there is no defined region (whole chromosome)

 

 

What is the role of telomerase?

 

it solved the "end" problem by prolonging special structures "telomeres" at chromosome ends.

 

 

DNA repair

What is a transition?

 

Purin versus Purin exchange

 

 

What is a transversion?

 

Purin versus Pyrimidin exchange, or vice versa

 

 

Name some sources of mistakes which could eventually lead to mutations:

 

tuatomeric structures of DNA bases

 

cell internal metabolies, e.g. reactive oxygen species

 

external chemicals e.g. alkylation agents

 

radiation e.g. UV or X-ray

 

deamination

 

 

What is a simple mechanism to repair UV induced tymine dimerization?

 

Direct reversal of the damage by photolyases

 

 

Ada is involved in alkylation repair, what was special about this repair

 

The protein transfers the alkyl group onto itself, as this is irreversible the protein is no enzymes and is thus "used up" . But it can still act as a regulator (see there)

 

 

What is the role of Mut SLH?

 

Its role is in mismatch repair MutS recognizes a mismatch, MutH a hemimethylated strand and nicks the strand; be prepared to explain the mechanism

 

 

Explain Base excision repair

 

damaged bases are removed by glycosylase (there are many different ones) AP site is recognized by an endonuclease, it cleaves the backbone and the hole is filled 3/17

 

 

When is nucleotide excision repair being used?

 

For bulky lesions

 

 

What are TLS polymerase

 

Translesion polymerases, they are able to polymerize in a non-templated fashion when damage has occurred

 

 

Why are TLS polymerase a last resort?

 

The TLs polymerase are error prone and just ensure that DNa can be replicated, this often leads to mutations. It can safeguard replication though ´

 

 

Explain the LexA-RecA system.

 

Normally LexA is blocking the syntehsis of SOS genes by binding to a DNA operator. If RecA bind ssDNA it is activated and cleaves LexA. Cleaved LexA can't bind to the operator anymore, SOS genes are read. =>2/27,28

 

 

Where do ATM and ATR play a role and what can activate them (in terms of which state not which protein)?

 

They play a role in the eukaryotic damage reponse e.g. to double strand breaks.

 

 

What are effects of ATR activation?

 

Cell cylce control, replication fork stabilization, replication origin control

 

 

Explain the regulation of p51.

 

See slide

 

 

Recombination

What is NHEJ

 

Non homologous end joining, joining DNA ends, it is predominant in non dividing cells. (G1 phase)

 

 

Explain the simplest case of NHEJ

 

A double stranded break is repaired by simply joining the ends potentially after trimming a few bases by a nuclease.

 

 

When resection occurs during NHEJ, what happens?

 

DNA ends are resected, regions of microhomology are found, DNA pairs, gaps are filled and overhangs trimmed

 

 

Briefly depict the process of homology directed repair with synthesis dependent strand annealing (no protein names are necessary, but give the neccessary terms for the DNA structures)?

 

Resection occurs to generate 3' overhangs, one of the 3' overhangs invades an intact duplex, generating a heteroduplex and a displacement loop,

 

new DNA is synthesized, until this can pair with the other 3' overhang. Then repair replication and synthesis (Probably helpful if you could draw a diagram here)

 

 

What is the role of RecBCD in E. coli?

 

It plays a role in homology directed repair, where it has nuclease and helicase activity and in the end generates 3' tails after encountering Chi sites.

 

 

What are Chi sites?

 

Special DNA sequences recognized by the recBCD complex.

 

 

Genes involved in repair and recombination are often implicated in some diseases as well, name two example for such a disease

 

Werner Syndrome

 

Bloom Syndrome

 

Rothmund Thomson Syndrome

 

 

What is the role of RecA (RAd51) in homology directed repair, where else does it occur?

 

It loads onto the single stranded DNA and helps in strand exchange it also plays a role in homologous recombination

 

 

What is the consequence of using homologous chromosomes as templates for homology-directed repair?

 

DNA is changed and is afterwards a copy of the homologous chromosome. This can lead to a loss of heterozygosity

 

 

Explain how yeast switches its mating type and why this is interesting for Molecular biology?

 

The switching uses homology directed repair. There are three loci the active MAT locus and two silent loci one carrying alpha and one a information.

 

The endonuclease HO cuts in the MAT locus and either the information of alpha or a is copied there potentially changing the mating type.

 

 

Does the cleavage of a holiday junction always lead to large areas of recombinant product?

 

No this depends on how the junction is cleaved

 

 

Where is RuvAB involved in the broad complex of recombination?

 

Movement of the holiday junctions

 

 

Depict the first steps in homologous recombination

 

After a double strand break, DNA is resected and bound by proteins which help in inavding the other duplex. DNA

 

is synthesized from the invading strand. The displaced strand of the D-loop is captured by the remaining 3' end.

 

DNA synthesis occurs and holiday junctions form 3/41

 

 

What is the relationship between homology directed repair and homologous recombination?

 

They are similar and the initial processes are almost the same

 

 

How can damaged replication forks (specifically nicks) be repaired with a principle reminiscent of recombination?

 

single strand nick leads to a ds break after strand separation. One strand of the "broken off" end invades the

 

"whole" part a D-loop structure is formed, DNA synthesis occurs, the displaces strand is captured by the lagging strand

 

lagging strand synthesis is restarted and after clevaged the fork is restored see

 

 

Describe Break Induced replication

 

Resection occurs to generate 3' overhangs, one of the 3' overhangs invades an intact duplex, generating a heteroduplex and a displacement loop,

 

new DNA is synthesized. The displaced strand from the D-loop is not captured. Here lagging strand synthesis starts occuring => 3/50

 

 

What are some consequences of Break Induced replication?

 

Potentially loss of heterozygousity

 

Potentially this Replication is not as precise as normal replication 4/58f

 

 

Name at least two problems that can occur with recombination due to sequence similarity regions?

 

Recombination on the same chromosome

 

Unequal crossover between chromatids or between homologs can lead to deletions and duplications

 

Crossing over between different chromosomes can lead to DNA translocation

 

 

Mobile Elements

Be ready to explain a C0t curve

 

See graph there

 

 

What did Barbara McClintock discover

 

transposition / transposable elements

 

 

Explain autonomous and non-autonomous elements

 

Autonomous elements encode the enzymes needed to move. Non-autonomous elements lack these and rely on enzymes of Autonomous elements

 

 

Would you rate the number of transposable elements in the human genome as high or low?

 

High

 

 

What is the state of many transposons in the human genome?

 

They are inactive e.g. due to mutation (also see first lecture)

 

 

If a Mobile Elements jumps into a functional gene, what can be the consequence for gene function?

 

The gene function or regulation can be affected potentially resulting in disease

 

 

Does transposition necessarily require homology?

 

No in general not, but be aware of the special cases

 

 

What are Class I and Class II elements?

 

Class I elements are retrotransposons that move via an RNA intermediate, Class II elements are DNA only transposons 5a/11

 

 

What is the general structure of a Class II element?

 

It has terminal inverted repeats and a transposase gene.

 

 

What are the two major Class I element groups?

 

LTR and non LTR

 

 

What is remarkable for Class II elements when it comes to the species barrier?

 

Class II elements can likely cross the species barrier

 

 

What is the predominant movement mechanism for DNA elements?

 

Cut and Paste

 

 

What is the less common movement mechanism for DNA elements?

 

Nick and Paste

 

 

When it comes to bacterial transposons, which other genes apart from the transposase do they often carry?

 

E.g. pathogenicity factors and antibiotica resistance

 

 

What is an IS Element?

 

A bacterial cut and paste DNA element only encoding its own transposase

 

 

What is a compound transposon?

 

Two simple transpons (IS elements) flanking (another) gene(s)

 

 

For a DNA cut and paste transposon, how many transposase proteins would you need in general to mobilize one transposon?

 

Two

 

 

How is it ensured that transposon ends are brought together before cutting for cut and paste DNA elements?

 

The transposase cuts in trans, i.e. they cut the other end that they don't bind

 

 

In the case of DNA cut and paste elements: When such an element inserts into the sequence CGAT. How will the sequence look after insertion.

 

GCATxxxxTransposonxxxxGCAT

 

 

After a DNA cut and paste transposon leaves a certain site a footprint remains. Explain what this is.

 

Upon insertion a piece of DNA is duplicated, when the transposon leaves, this duplicated DNA can remain -> a footprint

 

 

After a DNA cut and paste transposon leaves a certain site a precise excision can occur. Explain what this is and name conditions.

 

When the transposon leaves and the resulting gap can be repaired by homology directed repair from a transposon free site, the original state can be restored and no footprint remains

 

 

Explain in broad, superficial terms where VDJ recombination occurs?

 

Antibodies joining of different pieces, more possible antibodies, several possible fragments for each V D J pieces

 

 

What is the relationship between VDJ recombination and transposons?

 

They could be related as there are certain similarites both in the enzymes as well as in the signal DNA sequences

 

 

What is the P element and what is remarkable about it?

 

A DNA mobile element. It was only recently introduced into Drosophila by crossing the species barrier.

 

 

Name an application in the lab for the Drosophila P element?

 

Using microinjection this is a way to make transgenic flies.

 

 

What is sleeping beauty and how was "she" awakened from "her" sleep?

 

This is a fish transpon which was no longer active. Using the consensus sequence the ancestral working state could be restored

 

 

What is sleeping beauty and how did "she" fall asleep in the first place?

 

This is a fish transpon which was no longer active. Due to mutations the inserted transposons became "dormant"

 

 

Name the two classes of RNA retrotransposons

 

LTR and non-LTR elements

 

 

What does LTR stand for in LTR (RNA) elements?

 

long terminal repeat

 

 

Draw an archetypical LTR element.

 

>LTR=== GAG, PR, IN, RT-RH ===LTR>

 

 

Within a LTR Retroelement you usually find coding regions for 4 proteins, name these?

 

INtegrase, GAG, PRotease, RT-RnaseH reverse transcriptase+ RNAse activity

 

 

You have a working LTR retroelement, when you compare that to a retrovirus which protein coding regions is missing?

 

ENV for extracellular stage

 

 

Describe how a LTR element multiplies and integrates into new regions?

 

 

What can happen when you have two LTR elements spaced a bit apart from each other with the intervening DNA?

 

It can get lost due to homologous recombination between the elements

 

 

What is the relationship between LTR elements and retroviruses?

 

Explain the additional elements retroviruses have

 

 

Do you rate the non-LTR element occurrence in mammalian genomes as absent/low/medium/high/very high?

 

very high

 

 

Why are RNA elements very often mutated?

 

Reverse Transcription is error prone. reverse transcription is part of the life cycle.

 

 

What are the typical elements for a LINE element

 

ORF1/ORF2 and polyA

 

 

What is the sequence requirement for an insertion of some non-LTR elements (e.g. LINEs) and why?

 

a stretch of Ts that pair with the polyA tail

 

 

How does DNA methylation affect IS10 transposition?

 

DNA methylation both prevents transposase expression and transposon end activity

 

 

In which cells is the Drosophila P element active and in which one is it not active?

 

It is active in germ line but not somatic cells.

 

 

What is the role of piRNA?

 

They block transcription of certain transposons.

 

 

Ty5 is a relatively "benign" element when it comes to gene mutations why?

 

It preferably inserts into heterochromatin

 

 

Explain what local hopping means for certain transposons?

 

If transposons preferably insert in a location close to the donor site.

 

 

What does CSSR stand for?

 

Conservative site-specific recombination

 

 

When it comes to CSSR recombinases which enzyme class do they fall into?

 

They are topoisomerases

 

 

Which two major classes of CSSR recombinases exist (hint name the AA)?

 

Serine and Tyrosine type

 

 

What is the difference between Serine and Tyrosine Recombinases when it comes to the intermediate form, describe both forms?

 

Tyrosine recombinases break DNA and form DNA-3P-tyrosine linkage, Serine recombinases break DNA and form a DNA 5P-serine linkage

 

 

Draw and explain the recombination of a serine resolvase.

 

See the sequence in the lecture

 

 

Draw and explain how the Cre recombination occurs (a tyrosine recombinase).

 

See the sequence in the lecture

 

 

What is the Cre enzyme and what does it do?

 

a (tyrosine) recombinase that recognizes lox sites.

 

 

For what can the Cre/lox system be used for?

 

To delete DNA between two lox sites, this is often used as a tool where Cre is expressed under a specfic promoter. After Cre expression, a piece of DNA is removed and for example a tissue specific knockout is made.

 

 

The bacteriophage lambda system has four sites that are named on DNA (hint two sites get converted into two other sites), name the sites?

 

attP, attB -> attL, attR

 

 

What is the Invitrogen Gateway system system building on (which molecular mechanism from which organisms)?

 

phage lambda recombination

 

 

Transcription

What are the major two differences between RNA and DNA?

 

RNA has U instead of T and ribose instead of deoxyribose.

 

 

Name the three major phases in transcription

 

Initiation, Elongation, Termination

 

 

Name the three major eukaryotic RNA polymerases and their primary role

 

RNA polymerase I (large ribosomal RNA), II (mRNA), III (tRNA and 5S rRNA)

 

 

What is special for plant RNA polymerases?

 

They have an additional polymerases (apart form I, II , III) to transcribe regulatory RNAs

 

 

When it comes to how much work RNA polymerases have, how would you rate the transcription of RNA Polymerase I+III 0/4< x <1/4< x <2/4< x <3/4< x <4/4 ?

 

3/4 <x< 4/4 or the main load

 

 

What can happen to the C-terminal domain of eukaryotic RNA polymerase II?

 

It can become phosphorylated

 

 

What does the bacterial sigma factor do?

 

It contacts the promoter

 

 

What are the major two bacterial promoter elements (that are close to the TSS) (for sigma70) (hint name them by position)?

 

-35 and -10 element

 

 

In which bacterial promoters do you find the UP element? (somewhat active, medium active, very active)?

 

very active

 

 

Do you know the consensus sequence of the -10 sigma70 E.coli element?

 

TATAAT

 

 

What is an anti-sigma factor?

 

Proteins binding to sigma factors and inhibiting their function

 

 

In S. typhimurium flagellum assembly an anti sigma factor is involved, can you say how this factor FlgM is later loosing its influence

 

It is exported through the incomplete flagellum apparatus

 

 

Eukaryotic promoters have a box very similar in sequence to the bacterial -10 element, how is this box called?

 

TATA box

 

 

Which protein do all thee eukaryotic RNA polymerases need for establishing the pre-initiaion complex?

 

TATA binding protein TBP

 

 

When it comes to transcription what is the hallmark of a closed complex?

 

RNA polymerase is in position and the DNA is not yet opened, then the complex of polymerase and promoter is called closed complex

 

 

When it comes to transcription what is the hallmark of an open complex?

 

RNA polymerase has opened up the transcription bubble

 

 

Interestingly, when RNA polymerase starts making RNA, a strange phenomena occurs resulting in short pieces of RNA, explain this?

 

RNA polymerase is not immediately working fully instead it enters cycles of abortive initiation resulting in short pieces of RNA being released. This is likely due to a loop of eukaryotic TFIIB and bacterial sigma factors extending into the polymerase

 

 

What is promoter clearance?

 

When the loop of TFIIB/sigma inserted into the RNA polymerase is displaced and the polymerase breaks away from the promoter

 

 

What happens to the CTD when eukaryotic RNA polymerase is converted into the elongating complex after promoter clearance?

 

It becomes phosphorylated

 

 

Describe a simple! experiment to show that abortive initiation takes place in vitro

 

Incubate RNA polymerase holoenzyme with radioactive nucleotides. Run products on gel and visualize with Xray film

 

 

To show that abortive initiation takes place in vivo special kinds of nucleic acids were used, name them and indicate why these were used?

 

locked nucleic acids (these are locked in a conformation more favourable for hybridization)

 

 

What is the approximate speed of RNA polymerase 0<x <10 x <100< x <1000< x <10000< x <100000 nucleotides per second?

 

10< x <100 (20-50 nt/s)

 

 

What is promoter proximal pausing?

 

When there is some pausing after only 35-50 bp have been synthesized by RNA polymerase

 

 

What are potential reasons for transcriptional pausing?

 

Short complementary regions in the nascent transcript forming hairpins or weak DNA-RNA hybrids

 

 

Which factors can relieve transcriptional pausing?

 

Elongation factors

 

 

Explain how transcriptional pausing may be relieved?

 

RNA polymerases reverses direction, most recently made RNA separates off from DNA, this protruding end can be chopped off

 

 

What is one of the first maturation steps that occurs to eukaryotic mRNA?

 

This is often capping, happening while RNA is still being made

 

 

What happens after capping to the eukaryotic RNA polymerase II CTD?

 

It is phosphorylated at additional serines

 

 

What is the role of the 5' cap for the mRNA?

 

It helps in nuclease degradation, elongation termination of transcript, mRNA processing, export from the nucleus and directing translation

 

 

What is the minimal structure incorporated into a cap?

 

7-methylguanine is lined via a 5'-5' triphosphate to the 5' end of the RNA. (In more complex eurkaryotes 2' O of ribose in the second and third base gt methylated) 6a/37

 

 

Describe schematically how capping proceeds?

 

removal of 5' phosphate (PPP-RNA -> PP-RNA), addition of GMP (GPPP-RNA), methlyation of guaanine m7GPPP-RNA)6a/38

 

 

What takes care of the nucleosome problem during transcription?

 

histone chaperons

 

 

What topological problem can transcription cause?

 

Changes in supercoiling

 

 

What is a (bacterial) intrinsic terminator?

 

A site where bacterial RNA polymerase terminates transcription without any additional factors

 

 

What are the two main features of a bacterial terminator?

 

An inverted repeat that forms a stem loop in the RNA, and a polyA (DNA) region, that leads to less stable AU base pairs

 

 

What is the bacterial Rho protein (in the field transcription)?

 

An enzymatic terminator

 

 

What is the difference between intrinsic and Rho dependent genes in terms of termination?

 

The former make a hairpin and have a poly A region the latter (Rgho) don't

 

 

Which eukaryotic RNA polymerase recognizes intrinsic terminators?

 

RNA Polymerase III

 

 

When you compare bacterial intrinsic terminators to eu. RNA polymerase III terminators what does the latter not need?

 

eu. RNA Pol III doesn't need the RNA hairpin 6a/46

 

 

What is eu. RNA polymerase II termination coupled to?

 

It is coupled to 3' end processing

 

 

What happens to most eukaryotic 3' mRNAs?

 

They get polyadenylated

 

 

Is the polyA tail of eu. mRNA added to the last based made by RNA polymerase II?

 

No, the nascent transcript is cleaved first and then the polyA tail is added.

 

 

Give the names of the two major model for eu. RNA polymerase II termination?

 

torpedo and allosteric model

 

 

What must a RNA transcription regulatory protein do?

 

It must specifically recognize its right regulatory sequence e.g. through a protein domain

 

 

Why can it be favourable to contact the major grove of DNA in terms of specificity?

 

In the major grove all bases can potentially be distinguished this is not necessarily the case for the minor grove

 

 

Which kind of amino acids would you expect to find in proteins that contact DNA and why?

 

positively charged amino acids, as the DNA is negatively charged

 

 

Give two examples of domains or motifs found in transcription factors?

 

helix-turn-helix, homeodomain, zinc finger....

 

 

Describe the procaryotic trp repressor system?

 

See the slides

 

 

The lac system depends on the input of both glucose and lactose availability. Name the promoter states (off/weakly on/strongly on) in dependence of glucose and lactose concentration?

 

low glucose, lactose available -> strongly on

 

low glucose, lactose not available -> off

 

high glucose, lactose available -> weakly on

 

high glucose, lactose not available -> off

 

 

A colleague of yours proposes that bacteria don't phosphorylate proteins. Can you prove him wrong by naming a signal transduction pathway?

 

The two component signal transduction pathway features a histidine kinase.

 

 

Can transcriptional regulation only occur in the initiation phase?

 

No it can also occur during elongation and termination

 

 

What is anti-termination?

 

When transcription termination is actively prevented

 

 

Which famous virus uses anti-termination?

 

HIV

 

 

What are riboswitches?

 

Riboswitches are RNA regions that can directly bind a small molecule that controls the RNA secondary structure, regulating transcription or translation

 

 

Explain how translation is coupled to transcription for the bacterial trp operon?

 

see 103 (you would need to explain that there are 4 blocks, where 3+4 form a terminator but there is a small ORF with lots of Trp causing eventual staalling making 2 pair with 3) ...

 

 

Small RNA

What can Dicer (and Dicer Like) do to double stranded RNA?

 

It cleaves it into short pieces

 

 

How can plants defend themselves against plant viruses?

 

siRNA -> silencing (Give some details about AGO, Dicer etc)

 

 

What is remarkable about siRNA duplexes?

 

They can spread between cells.

 

 

What is Co-suppression?

 

When a construct is brought into a genome in normal sense orientation and both the introduced gene and the endogenous gene are silenced 7/29

 

 

You want to suppress a gene in C. elegans, which RNA would you introduce a) sense -----> b) antisense <-------- c) double stranded <-====->

 

double stranded

 

 

Can small RNAs effect DNA?

 

Yes siRNAs for example can target regions for Cytosine methylation or histone modifications

 

 

How many RNA polymerase classes would you find the genome of an angiosperm plant?

 

five

 

 

What is the role of RNA Pol IV in plants?

 

Production of siRNA

 

 

Describe the core components of how miRNA are processed in animals?

 

See slide: RNA Pol makes pri-mRNA which is cleaved into pre-miRNA (Drosha), exported from the nucleus (Exportin), there pre-miRNA is cleaved by Dicer and finally RISC complex is formed (what is not shown there is strand selection... just fro your info)

 

 

What is a *-strand?

 

The strand of a miRNA that is usually (or probably better: more often) degraded

 

 

Which gene class is often targeted by conserved miRNAs?

 

Transcription factors

 

 

RNA and then

Which kind of processing steps can eukaryotic RNA encounter?

 

Cleaving, splicing, polyAdenylation, Capping, Editing

 

 

Is the half-life of all RNAs similar?

 

no

 

 

What effects RNA stability?

 

5' and 3' structures (e.g. cap) hairpins, splicing

 

 

Explain bacterial/eukaryotic RNA degradation?

 

See slides

 

 

Which nucleotide is used by EF-TU/Ts?

 

GTP

 

 

Which trick to tranlsation factors often use to access the ribosome interface?

 

They can mimic tRNAs

 

 

Apart from a start codon which other sequence do you need in bacteria to start translation?

 

The Shine Dalgarno sequence (be prepared to explain how it works as well)

 

 

What is the Kozak sequence?

 

The Sequence A/GXXAUGG which is helping in recognizing the "right" AUG in eukaryotes

 

 

What is the closed loop complex in the topic of translation?

 

See corresponding slide

 

 

Is stop codon recognition sloppy?

 

No it ids highly accurate

 

 

what is tmRNA used for, where is it used and explain how it is used?

 

bacterial quality control, in bacteria, see slide

 

 

Explain the 21st amino acid and its protein incorporation?

 

Selenocytstein, see slide

 

 

Is Pyrrolysine a proteinogenic amino acid?

 

yes

 

 

What is the magic spot?

 

(p)ppGpp which is synthesised through RelA

 

 

Give an example for the less common 5' UTR regulation in eukaryotes?

 

ferritin : be prepared to explain the mechanism

 

 

Give an example for the less common 5' UTR regulation in eukaryotes?

 

ferritin : be prepared to explain the mechanism

 

 

How is it achieved that in X.laevis oocytes mRNA is initally not translated?

 

Explain the mechanism in general!

 

 

How is splicing coupled to NMD?

 

Explain the EJC and where you would normally encounter splice sites and how this can be used to find premature stop codons

 

 

Folding

Explain roughly how Hsp70 chaperones work?

 

These prevent hydrophobic patches to aggregate

 

 

Explain how GroEL/ES chaperones work?

 

GroEL binds, later GroES a switch occurs exposing different hydrophilic/hydrophobic regions, protein gets refolded

 

 

What is PDI?

 

Potein disulfide oixidase, it both oxidises and swaps disulfide bonds

 

 

In which diseases do prions seem to play a role (at least two)?

 

BSE, Scrapie, nvCJD, GSS ....

 

 

Are prions only occuring in higher organisms?

 

No yeast also has prions

 

 

Can you cure certain prions and if yes how?

 

Certain yeast prions can be cured by chaperons

 

 

Explain what inteins are and their potential application in biotechnology?

 

Protein regions which autocatalytically "splice" themselves out of the protein. They can be used in chemical semi-synthesis

 

 

Name the three main types of lipd modification?

 

Acylation, Prenylation, GPI anchors

 

 

What is interesting about hedgehog?

 

It is related to inteins and it is modified with cholesterol

 

 

If you add carbohydrates to proteins how does this often effect these?

 

They can become more soluble and spacious

 

 

What can ROS and NOS lead to?

 

modified AA, give at least on example each

 

 

Depict the UBQ pathway?

 

see slide you must include E1, E2, E3

 

 

Looking at the UBQ pathway, how many isoforms do you have of the respective enzymes?

 

Often one E1, several E2 and very many E3

 

 

If you attach polyubq via K48 what does it lead to?

 

degradation in the proteasome

 

 

What does the proteasome do?

 

degrade (certain!) ubq marked proteins

 

 

Explain one unfolded protein response (ATF6) in eukaryotes?

 

see slide

 

 

Sorting

Explain one unfolded protein response (sigmeE) in prokaryotes?

 

see slide

 

 

Explain how proteins get imported into the ER via the SRP?

 

see slide: recognition of sequence/ribosome stops/SRP-ribo gets recognized by SRP-receptor/peptide chain moved to translocator

 

 

What is the SRP composed of?

 

protein SU as well as 7S RNA

 

 

How are proteins brought into/out of the nucleus?

 

importins/exportins

 

 

How do importins/exportins "know" when to release their cargo?

 

via GTP /see slides for the cylces

 

 

Why can import into chlorplast/mitos complex?

 

Multiple compartments that need to be targeted : thus TIM/TOM TIC/TOC

 

 

Why can import into chlorplast/mitos complex?

 

Multiple compartments that need to be targeted : thus TIM/TOM TIC/TOC