Genetics exam 3

Examples of noncoding RNA

mRNA, rRNA, tRNA

What are the major differences b/w RNA and DNA

RNA has a 2’OH group, RNA uses Uracil instead of Thymine, RNA is single stranded

RNA can fold on itself (hair pin and clover etc.)

What does the 2’OH group in RNA add (chemically/structurally)

structural flexibility
decreased stability through hydrolysis

Uracil

used rather than thymine
can still pair w/adenine (2 H-bonds)

can also pair w/guanine (2-H bonds)

Why is it advantageous to have a different base in DNA vs RNA

Uracil is cheaper to make energetically for a short-lived molecule
cytosine can deaminate to uracil in DNA, so mechanisms have evolved to eliminate uracil point mutations in DNA (where long-term stability is needed)

Transcription

copying the code from DNA to RNA

Which strand do RNA nucleotides base pair to?

the template (antisense) strand

Which strand is the RNA sequence identical to (RNA uses U rather than T)

the coding (sense) strand

Gene sequences are written using the?

sense strand

RNA is transcribed in the?

5’ to 3’ direction, just like DNA replication

Genes can be transcribed from?

both strands of DNA, either can be template or coding depending on gene

RNA polymerase adds rNTPs to the?

3’OH group.

mechanism very similar to DNA replication

promoter

site of RNA polymerase binding

Transcription start site (TSS)

first transcribed base

CDS

coding sequence

UTR

untranslated region (5’ and 3’)

initiation of transcription

RNA polymerase binds to promoter and opens the DNA duplex

Elongation of transcription

RNA polymerase adds bases to 3’ end of lengthening RNA

Termination of transcription

RNA polymerase dissociates from DNA template and stops adding bases

Transcription initiation in bacteria

The single RNA polymerase holoenzyme transcribes RNA from DNA template
- DNA dependent RNA polymerase
- 6 polypeptides: 5 in core enzyme + sigma factor

What does sigma factor do?

• directs RNA pol to the promoter and orients it properly

• helps “melt” the DNA duplex to form an open complex

• dissociates from RNA Pol after transcription begins

  • 7 sigma factors in E.coli

Promotor sequences in E. coli

• Sextama box - TTGACAT

• Pribnow box: TATAAT

Transcription elongation in bacteria

• RNA is synthesized in the 5’ to 3’ direction

  • RNA Pol moves from 3’ to 5’ on the template strand, unwinding and rewinding DNA

  • New NTPs are added to the 3’ OH of the existing RNA polymer

  • The 5’ end of the RNA is extruded from the RNA polymerase

  • Energy to drive RNA synthesis comes from the hydrolysis of the NTPs

Transcription termination in E.coli

• Factor-independent termination

• Rho-dependent termination

Factor-independent termination

• Termination signaled w/in the RNA sequence

  • RNA pol pauses at A-rich region

  • RNA pol reverses to stabilize duplex

  • RNA pol encounters the GC-rich stem loop RNA (hairpin)

  • This releases RNA from RNA Pol and RNA Pol from DNA

Rho dependent termination

• Termination mediated through the helicase Rho

  • Rho binds to the upstream Rho utilization site (rut site) in RNA

  • Rho helicase travels towards the 3’ end of transcript

  • When Rho encounters a paused RNA Pol, Rho unwinds the DNA-RNA duplex and releases RNA from RNA Pol, ending transcription

Endonucleases

cuts DNA in the middle

Exonucleases

Cuts DNA from end

RNA decay in bacteria

• pyrophosphohydrolase removes a pyrophosphate (PPi), leaving single phosphate at 5’ end

• RNase E (endonuclease) binds to single phosphate, chews up DNA

• Exposed ends of RNA can now be degraded by exonuclease present in cell

Gene expression in Prokaryotes

• DNA is not compartmentalized

• RNA is transcribed via RNA polymerase

• Translation occurs immediately after transcription (co-transcriptionally)

• genes are packed tightly together on chromosome

Gene expression in eukaryotes

• DNA compartmentalized in nucleus

• RNA transcribed via RNA polymerase

• RNA is processed before nuclear export

• Translation occurs post-transcriptionally in the cytoplasm

• genes are often spaced far apart, with lots of intergenic DNA sequence

The 3 RNA polymerases that transcribe eukaryotic RNAs

• RNA polymerase 1: most rRNAs (except 5S rRNA)

• RNA polymerase 2: mRNAs, some noncoding RNAs including microRNAs (miRNA), long noncoding RNAs (IncRNAs), and some small nuclear RNAs (snRNAs)

• RNA polymerase 3: tRNAs, 5S rRNA, and some snRNAs

Transcription initiation of RNA Pol II

• Generic Transcription Factors (GTFs) are proteins that bind to promoter elements and recruit RNA polymerase

  • TATA box (-35) in only 25% of human genes

• The TFIID is the first GTF to bind to the promoter

  • TBP = TATA binding protein

  • TAF = TATA binding associated factor

• Once bound, TFIID recruits other GTFs and RNA polymerase II to form the Pre-initiation complex (PIC)

  • GTFs position RNA pol II at the transcription start site and unwind the DNA to form a transcription bubble

Transcription elongation of RNA pol II

• During elongation, RNA Pol II and many GTFs dissociate, through TFIID remains to recruit another RNA Pol II molecule

• RNA Pol II often pauses approximately 50 bp downstream of the promoter

  • NELF (negative elongation factor) and DSIF (DRB sensitivity-inducing factor) promote pausing

  • phosphorylation of both NELF and DSIF by P-TEFb (positive transcription elongation factor) unpauses RNA Pol II

The CTD (C-terminal domain) of RNA pol II is modified to?

• interact with different processing factors

  • The CTD consists of repeating subunits of the amino acid sequence YSPTSPS

  • select amino acids (e.g. serine) can be phosphorylated via kinases + ATP

  • Variable phosphorylation of serines in the CTD results in binding to different processing factors as elongation proceeds (capping and polyadenylation factors)

RNA processing 1: a methylguanosine (m7G cap) is added to the 5’ end of the RNA

• the emerging 5’ end of the RNA transcript interacts w/capping enzymes bound to the CTD of RNA Pol II

Purpose of the m7G (5’ cap) cap

• protects the RNA from degradation by exonucleases

• is a binding site for proteins involved in RNA processing and nuclear export

• necessary for translation: the cap binds to the other proteins that recruit the ribosome

RNA processing 2: RNA transcribed by Pol II is cleaved and polyadenylated

• CPSF (cleavage and polyadenylation specificity factor) bind to an AAUAAA sequence in the 3’ UTR

  • CPSF directly cleaves the RNA molecule (15-30 nt downstream of binding site)

  • CPSF recruits poly(A) polymerase (PAP)

Purpose of polyadenylation (adding the Poly(A) tail)

• Protects the RNA from degradation by exonuclease through interactions with the poly(A) binding proteins (PABPs)

• interacts with translation factors, also through PABPs

Transcription termination of RNA Pol II

• Torpedo termination model

• allosteric termination model

Torpedo termination model

• After cleavage and polyadenylation, the 5’ to 3’ exonuclease Xrn2 chews up the strand of RNA still bound to RNA pol II

• Xrn2 makes contact with RNA pol II, knocking it off the DNA

Allosteric termination model

• dissociation of elongation factors after cleavage results in allosteric changes to RNA pol II - largely unknown mechanism

• reshaped RNA pol II dissociates from DNA and RNA

Splicing removes?

introns from pre-mRNA and splices together remaining exons

Alternative splicing can?

• produce multiple mature RNAs and proteins from a single gene (isoforms)

  • in humans, over 80k proteins are produced from only 20k genes

  • alternative splicing allows for increased diversity at protein level

Spliceosome

• binds to the CTD of RNA pol II

• made up of 5 snRNPs

• The snRNAs base pair to the intronic RNA and catalyze the splicing reaction

Splicing is regulated through 3 consensus sequences in/near introns

• splice donor site:5’ GU is universal

• splice acceptor site: 3’ AG is universal

• branch point: A is universal

spliceosome assembly

• U1 snRNP binds the 5’ splice site; U2 snRNP binds to the branch point

• U4, U5, and U6 snRNPs join the spliceosome as a preformed complex

• U1 and U4 snRNPs are released from the spliceosome

• U6 snRNP binds the 5’ splice site

• first catalytic step

• second catalytic step

• lariat intron formation + linked exons

Exons are linked together via

OH groups attacking phosphate groups to form new esters (part of overall spliceosome assembly process)

Eukaryotic RNA decay pathways typically start with?

exonuclease, not endonuclease

RNA interference (RNAi) can also result in?

• targeted degradation of RNAs

  • short double stranded noncoding RNAs trigger degradation of complementary RNA sequences

RNA polymerase binds to ______ to transcribe downstream genes

promoter

Bacterial genes are often linked in an ______ - genes are transcribed together in a single mRNA

• operon

  • genes in the same operon often perform similar functions or function in the same process

Genes can be turned?

on or off through gene regulationg

gene regulation

the process that controls the timing, location, and amount of gene products (RNA or protein) that is produced by a cell

The lac operon

• expresses genes necessary for the metabolism of the sugar lactose

  • Z, Y, and A

  • Z produces beta-galactosidase

  • Y produces permease

  • A produces transacetylase

B-Galactosidase (B-Gal)

• cleaves lactose into glucose and galactose

• modifies lactose into allolactose per

permease

transports lactose into cell

transacetylase

not required for lactose metabolism

P-

RNA polymerase cannot bind to promoter

Z-

lac Z loss of function allele (no B-gal activity)

Y-

lacY loss of function allele (no permease activity)

If the genotype is P+Z+Y+ will lac Z and lac Y be expressed?

yes

B-gal can cleave the chemical x-gal into?

galactose + blue precipitate

Would you expect to see white or blue colonies in E. coli grown in media supplemented w/x-gal for p-Z+Y+ w/out lacZ? P+Z-Y+ w/out lacZ?

White, white

What is the first problem when it comes to the lac operon?

how to ensure lac operon genes are expressed only when lactose is present

How does a bacterial cell ensure that the lac operon genes are expressed only when lactose is present?

• a repressor binds to the lac operon and turns off gene expression in absence of lactose

  • when no lactose is present, the lac repressor binds to a DNA element downstream of the promoter called at the operator (O)

  • when bound to the operator, the lac repressor blocks RNA polymerase from proceeding down the DNA

  • The lac repressor is expressed from a nearby one I, which is not part of the lac operon

  • repressor has allosteric and DNA binding site

How many sites can be active at one time on lac repressor?

one

Allosteric binding

• conformational change in lac repressor, inactivating DNA binding site

  • B-gal converts lactose into its isomer allolactose, so the repressor will not bind to the operator in the presence of lactose

  • this is an example of negative regulation - gene expression is blocked by a repressor

White or blue; I+O+P+Z+Y+? I-O+P+Z+Y+? w/and w/out lactose

white, blue

blue, blue

F’ plasmid with I repressor genes or lac operon genes can be introduced into an?

F- cell

F’/F- > I+O+P-Z+Y+/I-O+P+Z+Y+ w/and w/out lactose

white, blue

cis regulatory elements

regulate only genes on the same molecule of DNA (O and P)

trans regulatory elements

regulate nearby or distant genes, as the produce diffusible molecules (I)

I+ is ______ over I-

dominant

Oc

• constitutively active operator

  • operator DNA is mutated so that repressor can never bind

  • downstream genes are always expressed (cis-acting)

Is

• superrepressor

  • allosteric binding site of repressor is mutated so that allolactose can never bind

  • super repressor always binds to O+ operators (trans-acting), so downstream genes are always turned off

Is is ______ over I+

dominant

Lac operon problem 2

glucose is much richer energy source than lactose, how does bacterial cell ensure that the lac operon genes are expressed only if glucose is absent?

glucose is much richer energy source than lactose, how does bacterial cell ensure that the lac operon genes are expressed only if glucose is absent?

• glucose regulates levels of cAMP (cyclic adenosine monophosphate)

• high levels of cAMP (low glucose) bind to CAP (catabolite activator protein) and recruit RNA polymerase to the lac promoter

• example of positive regulation - gene expression is induced by an activator

In prokaryotes, the default state for most genes is? Eukaryotes?

on, off

TF (transcription factors) bind to DNA to regulate?

• gene expression

• Generic transcription factors (GTFs) bind directly to the core promoter DNA to recruit RNA polymerase

Do transcription factors bind to cis or trans regulatory elements?

Cis regulatory elements. Transcription factors bind to specific DNA sequences known as cis-regulatory elements to regulate gene expression.

Transcription factors bind directly to upstream ______ to regulate gene expression?

• Enhancer

  • enhancers can be proximal (nearby) or distal (far away)

  • repressors bind to cis-regulator elements sometimes called silencers

structure of transcription factors

• DNA-binding domain (DBD)

• activation domain (AD) or repression domain (RD)

  • sometimes: dimerization domain and ligand-binding domain (especially hormone receptors)

Distal enhancers loop around to interact w/core promoter via TFs and _____?

• mediators

  • mediators do not directly bind to DNA

  • can be coactivators or corepressors

How is cis-regulatory element (enhancer) activity detected?

• reportersare used to measure gene expression levels, often through the use of fluorescent or luminescent tags.

DNA analysis of loss-of-function mutations can identify important ________?

• cis-regulator elements

• point mutations coupled with a reporter

In yeast, how does the gal pathway regulate metabolism in the presence of galactose?

• Gal 1, 2, 7, and 10 are all turned on in presence of galactose and absence of glucose

• eukaryotic genes are typically not structured as operons - each gene has its own promoter

• Gal 1, 2, 7, and 10 all share the same enhancer > UAS (upstream activation sequence)

Gal 4 (a TF)

• always turned on in yeast cells

• binds to UAS enhancers to activate transcription

Gal80 and Gal3

• Gal80 and Gal3 are constitutively expressed

  • Gal80 is a co-repressor, Gal3 is a co-activator when bound to galactose

  • Gal80 binds to Gal4 AD, inactivating Gal4

  • in presence of galactose, Gal3 shape changes and binds to Gal80

  • binding of Gal3 to Gal80 results in shape change and dissociation from Gal4

TFs are ____ in nature, w/each subunit able to function independently

• modular

  • Gal4 DNA-binding domain can bind to UAS in absence of activation domain

  • Gal4 activation domain can be fused to an alternative DBD and still function to activate transcription

The Gal4/UAS system can be used to drive expression of ______ in other eukaryotes

• trans genes

  • crossing a Gal4 driver fly to a UAS-target fly results in progeny w/both Gal4 and UAS-target

  • approximately 10,000 Gal4 drivers available in drosophila stock

  • enhancers can be specific to cell type, tissue, developmental stage, or environmental trigger

Eukaryotic DNA is packed with proteins called______ into chromatin

• histones

• DNA in chromatin is compacted over 10,000 fold compared to unpackaged DNA

DNA wraps around an _____ or core histones to form a nucleosome

• octamer

  • 2 copies each of core histones H2A, H2B, H3, and H4 make up the octamer

  • a fifth histone, H1, is a linker histone that binds DNA that links histones together

  • 146 bp of DNA wraps around histone octamer approximately 1.7 times

  • flexible histone tails (N-terminus) are involved in interactions with other proteins and neighboring nucleosomes

  • histones are rich in Basic (positively charged) amino acids: lysine and arginine

  • the positive charge in histones allows them to tightly associate with negatively-charged DNA

Gene regulation by chromatin structure

transcription is unlikely to occur when DNA is highly compacted into heterochromatin but is possible in more open euchromatin

nucleosomes are excluded from enhancers and promoters of _____ genes

• active

  • nucleosome-free regions at enhancers and promoters are flanked by nucleosomes

  • if an enhancer/promoter are wrapped = no expression

insulators can ____ enhancers from interacting with/nearby promoters

block

Insulators can interact with each other to form?

• TADs (topologically associating domain)

  • insulators interact with each other through the DNA-binding protein CTCF

  • the loop of DNA bound by insulators is known as TAD

  • DNA sequences w/in a TAD preferentially interact w/each other

Two major mechanisms to make DNA accessible

• chromatin modification: alteration of the DNA and/or histones to affect recruitment of transcription factors

• chromatin remodeling: histones are repositioned or removed to allow access to promoters and enhancers

Acetylation of histone tails

• chromatin modification

• loosens interaction w/DNA

• acetyl groups bind to and neutralize the positive charges of lysines, loosening the interaction between histones and DNA

• acetyl groups added via histone acetyltransferase (HAT) and removed via histone deacetylace (HDAC)

• TFs containing bromodomains will bind to acetylated histones, promoting transcription