BIOB11 Final Exam (7-12)

General structure of AAs

has an

amino (N terminus) and carboxyl end (C terminus)

and a side chain (or R

group)

General structure of Polypepetides

linear sequence of AAs linked by peptide bonds

General structure of Subunits

made of domains, and can bind together to form proteins. (stable on their own also)

General structure of Proteins

composed of 1 or more polypeptide subunits. folded into correct shape and have correct post translational modifications to be functional.

what is a protein domain?

structural units of proteins (one blobby), can have the same ones in different proteins.

they fold independently and can have different functions.

what promote protein folding?

non covalent interactions

two ways duplication of a domain can occur?

exon shuffling and gene duplication

what kind of bond occurs in glycosylation?

covalent

What major step is the ribosome put into the correct reading frame during translation, and how?

Initiation, by reading the start codon, AUG methionine

explain,

the three sites of association with trna in ribosomes. trna enters in A.

between which two parts of the trna/AA does the peptide bond form ?

C-terminus of growing chain (the growing end) connects to the amino group of the new AA.

Explain translation elongation (5)

1. new tRNA comes in

2. peptidyl transferase activity results in bonding of chain and new AA.

3. L subU slides, A is empty

4. S subU slides to catch up.

5. empty trna(in E space) ejected

Explain what Translation elongation factors do. ex: EF-Tu and EF-G in

bacteria

• improve efficiency and accuracy

• holds the AA away from chain, until its sure its the right one

• if approved, GTP is hydrolyzed to GDP and then the TEF is released, allowing the AA to bond.

Adding AA to chain is irreversible. T or F

T, thats why we have the accuracy checks by TEFs

in both eukaryotes and prokaryotes, translation can begin at the 5’ end while

RNA polymerase is still synthesizing T ir F

F, only Proks because the rxn is coupled, but in eukaryotes cannot because it is not coupled.

the 5’ end of a PP chain is always methionine T or F

T, but it can be removed later on.

In prokaryotes, the small ribosomal subunit binds to the first AUG codon guided by a specific

sequence of nucleotides upstream. it is complementary to a sequence near 3’ end of 16S rRNA →

positions the ribosome at the correct spot

The Shine-Dalgarno Sequence (5’-AGGAGG-3’)

• they allow prokaryotes to synthesize multiple proteins from a single RNA

IF1, IF2, and IF3 do what?

IF1: attachment to mRNA

IF2: GTP-binding protein, attachment of first AA-

tRNA

• IF3: Prevents premature attachment of large subunit

• when they detach, L subU can bind=elongation occurs

what is N-formylmethionine (fMet)

In bacteria the initial AUG methionine is a modified

version

• Initiator tRNA (carrying fMet) interacts with AUG

  at what will be the ‘P’ site of the ribosome

eukaryotes have larger ribosomes than prokaryotes T or F

true! thats why their initiation is a bit more complex.

eIFs bind to the small subunit and are important for:

eukaryotic translation initiation.

eIF2, eIF1, eIF3 roles

euk TL initiation, encounters mRNA in ribosome:

• Initiator tRNA (Met) binding the ‘P’ site with GTP-bound eIF2

• eIF1s: Conformational change to allow binding of mRNA

• eIF3: interaction with eIF4G on mRNA complex

eIF4E/A/G roles in euk TL initiation

the set that belongs to the mRNA

E: binds to 5’ cap

A: unwinds any doub.stranded regions in the mRNA by ATP hydrolysis

G: links both ends to make a circular message/ (interacts with eIF3 on S subU)

Kozak sequence

S ribo subU with eIFs and initiator tRNA scans from 5’ end to find this consensus sequence to begin TL initiation

how are eIFs released, and what does that occur after

eIF’s GTP hydrolyzed and they are released. this allows for L subU to attach.

inefficient kozak sequence recognition results in:

P with different AA at the beginning (N terminus)

release factors bind to the A section and catalyze the addition of what instead of an AA at a stop codon?

water!

this frees up the C terminus. po

polysome

Multiple ribosomes associated with an mRNA

can occur anywhere in proks and only in cytoplasm in euks.

at at what point can another ribosome assemble?wh

when the AUG is free

differrential gene expression evidence

• some genes are transcribed more than others.

• in a drosophila embryo, in situ hybridization, which detects mRNA through base-pairing

  with a labeled nucleic acid probe , they saw odd-skipped mRNA. diff mRNA expressed in diff. cell types in a v precise manner,

genomic equivalence and evidence

all somatic cells of an organism have the same DNA, possible because of differential gene expression.

proof: An entire mammal can be cloned from the DNA in an adult somatic cell!

3 different levels of commitment/potential in stem cells

Pluripotent – Produce all cell types in the body.

(ex. embryonic stem cells)

Multipotent – Produce a related group of cells.

(ex.hematopoietic stem cells that create red and white blood cells)

Unipotent - Only produce cells of their own type, but have the property of self-renewal required to be labeled a stem cell.

(ex. muscle stem cells).

what is cell commitment

the gradual process of a stem cell becoming differential and functional (restricting cell fate)

what are the amplification steps in P expression, why?

one gene can be multiple mRNAs and 1 mRNA can become many proteins.

what is this? what des it show?

2D gel electrophoresis. separates proteins by mass and charge. shows different proteins expressed in different cells.

• general approach & potential uses for RNA-seq

USE: comparing transcriptomes of 2 diff tissues/ treatments, detect new muts or splicing variants.

HOW: isolate [ALL] mRNA, rev.transcriptase to → cDNA, ligate&amplify, sequence by synthesis

results in a sequence read. shows how many times a gene comes up.

what is this?

RNA-seq by heatmap

done for large scale comparisons of transcriptomes or proteomes.

colour scheme used to show different expression groupings.

in RNA seq. why are introns detected a little bit?

they are possibly not fully processed rnas (introns not spliced out yet) or some bits are kept for different functions.

Is protein binding to DNA typically using covalent or non-covalent bonds?

non-covalent

cis regulatory transcriptional factors

promoters and enhancers

On the gene they affect

on DNA, so same for all cells of an organism.

trans regulatory factors

ACTIVATORS OR REPRESSORS (and transcription factors)

bind to cis reg TC factors

expressed from anywhere in the genome

what is this, what is purpose

Mediator complex, Transcription regulators act through them. but not all, some interact directly with with

the general transcription

factors or RNA polymerase

or chromatin remodelers!

Transcription factors (TFs)

proteins that bind specific DNA sequences and influence transcription

Whether a TF can increase or decrease transcription is often dependent on forming complexes with other trans-regulatory elements.

explain the modularity of TFs (3)

• 1 gene, ++TFs

• 1 TF, ++genes

• 1 TF can have ++ effects in diff contexts.

what’s a way to create the opportunity for fewer proteins to be used to recognize a greater number of sequences

Dimerization: diff combos!

Genome-wide analysis of the binding locations/sequences for a particular protein (determining its fxn) can be performed by a technique known as:

ChIP-Seq

(chromatin immunoprecipitation and

sequencing)

which are chip seq and which are RNA seq.

top three chip seq, last one RNA seq

• general approach & potential uses for ChIP-Seq

1. crosslink uses covalent bonding to freeze all activity in the cell

2. DNA is broken into pieces, so only the target DNA is stuck to the select protein

3. antibody attaches to all select P and pulls out all DNA fragments that are attached to it.

4. reverse cross linking then sequencing possible!

Detects regulatory sequences

because enhancers are cis, they must be close to the transcription start site. T or F

F, because DNA can loop around!

promoter role

Sequences of DNA where RNA

polymerase can be recruited to

initiate transcription

enhancer role

Sequences of DNA that impact

how much gene product is made

from a promoter

if all cis regulatory genome is the same in every cell type, what differes/ ?

the combo of transcription factors and co-regulators present!

Explain modularity of gene regulation

1 gene, ++enhancers

1 enhancer, ++cell types, time, stimulus

• Explain how reporter genes can be used to understand regulation of a gene

• find and study enhancer sequences

• expression in DNA fused with reporter genes is visible.RG ex: Green fluorescent protein

  (GFP)

• allows for deletion mapping

what is deletion mapping?

delete a section, does anything change?

if the reporter gene fuses into one after change, it means one stripe of expression requires this one regulatory

sequence

WHATS A silencer?

cis regulatory element that can prevent promoter use and inhibit transcription.

Required to restrict gene expression to the proper cells/times

ex: NRSE sequence to the LacZ gene

activators usually interact with rna polymerase either directly or through mediators and GTFs. t or F

T, in proks though, more likely direct contact. according to chat GTP)A

Activators bind to

enhancer DNA

elementsr

repressors bind to

silencer Dna elements

Co-activators and

co-repressors are

recruited to help

influence

transcription

• Explain how nucleosomes impact DNA binding and contribute to gene regulation

if the surface the TF binds is wound around nucleosome, it cannot bind. some TFs can destabilize nucleosomes to facilitate binding.

note: epigenetic stuff happening parallel to. this

activators can bind to histone acetyltransferases, what does this do?

opens up chromatin and promotes gene transcription

Activator vs repressor when they are overlapping!

repressor winds, TCrepressed

if Activator and repressor are side by side, what happens

they stop each other (I think, according to slideh) (or repressor wins according to chat)

how does the recruitment of chromatin remodelling completes affect TC

repress TC because it re curls up the chromatin

how does the recruitment of histone deacytelyases or histone methyl transferees affect TC

repress TC as it promotes constriction tightness of the chromatin

why is lncRNA kinda special…? 👉👈 3 things

• non protein regulator

• can be cis or trans(normally)

• can start acting when its still being transcribed on

how are gene expression patters maintained in daughter cells?

Histone reader-writer complexes and DNA methyltransferases propagate and maintain epigenetic regulation of chromatin

If enhancers and silencers can be located far away from a promoter (and trigger chromatin modifications) what stops them from acting on the wrong gene?

Insulators (& the proteins that bind tothem) can alter the 3D arrangement of chromatin to divide DNA into looped region. only looped bits can have things happen.

how is coordinated expression off gene groups possible? (in development or in response to external stimuli)

1 transcriptional regulator can act with many different genes. ex: 1 hormone released connects to many different receptor Ps. allows for coordinated regulation and expression!

“Negative and positive feedback

loops can assemble into

complex gene regulatory

networks for precise control of

gene expression” Euks or Proks?

euks

benefits of operon regulation in prokaryotic genes.

Changes in gene expression occur rapidly in response to changes in nutritional and physical

environments → allows effective use of available resources

in prokaryotes: Polycistronic clusters of genes (an operon) share cis-regulatory elements (operator or activators or both) T or F

true! ex: Lac operon

how can mechanisms in prokaryotic gene expression be used in designed expression systems?

not sure…

why can genes only be regulated by a limited number of diff transcription factors?

[cause there are so many different types of combos and stuff… allowing for lots of variation?] there’s no need?

• regulation of transcription vs. post-transcriptional regulation

reg of TC: the RNA made from DNA

post TC: influences how much P is made, anything happening on the RNA live;

why can some Alt splicing be constitutive

(euks only btw!)

happens when the 5’ and 3’ splice sites are not clear, results in diff Ps, versions, which can sometimes be useful!

a repressor on a splice site is an example of what kind of regulation?

Negative control as access to splice site prevented= no splicing

positive control=activator

the presence of what marks the completed splice?

Exon junction complexes (EJC)

T or F: enhancer[cis sequences in general?] will always be present, its just a matter of whether or not trans regulatory factors will bind.

T

Nonsense-Mediated Decay:

there are in-frame stop codons in introns, if they are not spliced out, ribosome can detect the stop codons and disintegrate the mRNA.

this is done by the Upf proteins which connects stop codon with EJC

RNA editing is controlled by:

whether or not enzymes that edit are expressed in that cell type

Alt cleavage and polyadenylation

fine tuning mechanism that occurs after processing. indicated by Cstf cleavage event.

low cstf=normal bit spliced out bc poly A site is strong.

bad stuff present=high Cstf= increased site of binding to weak site (different length mRNA)→ antibodies secreted

(affects C terminal of P)

Three main types types of control acting through UTRs

1. mRNA stability (half-life of mRNA determines how long the message is

present to be used for translation)

2. mRNA translation (yes/no, when, how often)

3. mRNA localization (where in the cell)

(mRNA stability) In the cytoplasm, _________ gradually shorten the poly-A tail until degradation of the mRNA is triggered.

deadenylases

BUT Sequences in the 3’UTR regulate recruitment of proteins that

regulate poly(A) tail length (Change 3’UTR = change mRNA half-life)

(mRNA stability) -nuclease often means

breaking down

(mRNA stability) endo vs exo nuclease activity

both degrade RNA as a way to regulate

endo: by cleaving, blocking or exposing this site can regulate degradation.

exo: degradation from the outside PolyA tail 3’ end.

(mRNA translation)in the example where cells regulate based on the Iron levels, what do ferritin do?

binds excess iron

aconitase is on 5’UTR (b4!)

(mRNA translation)in the example where cells regulate based on the Iron levels, what do transferrin do?

imports iron into the cell.

aconite is on 3’UTR(after!)

(mRNA translation)what happens when iron starvation? in cytosolic aconitase

stays bound, so ferritin not activated, and transferrin activated, iron is transferred into cell

the opposite occurs in iron surplus, where the iron binds to aconitase and it releases. this allows transferrin 3’ poly A tail to degrade slowly, while ferritin is activated.

(mRNA translation)blocking the shine dalgarno sequence blocks TL in eukaryotes or prokaryotes

prokaryotes

Can also sometimes be affected by temperature!

(mRNA localization) Regulation of localization typically occurs through regulators binding to

sequences in the 5’ and 3’ UTRs T or F

T

(mRNA localization)

getting stuck to certain P

(mRNA localization)

protection, only the ones stuck are safe

(mRNA localization)

protein highways with motor proteins that walk along it carrying RNA

why is the mRNA localization important? (2)

localized translation

possibility of unequal distribution of daughter cells ( notable in development and stem cell differentiation)

Ex: neurons are so long!

Maternal contributions

proteins and RNA that are stored in the egg until they are needed to orchestrate early development (and determine the fate of particular cells). then real DNA takes over for later stages.

P-bodies role

sites or mRNA degradation or storing Translationally repressed mRNA.

mRNA that has been stored in a P-body can also be moved back to the cytoplasm

to reactivate translation T or F

true, bc there is not a membrane around these regions

what are stress granules?

Under conditions of stress, cells accumulate mRNA in another membrane-less compartment called stress granules

• In stress granules, translation initiation is blocked until the stressful conditions are removed