Module 3 Notes

DNA

A double-stranded molecule that forms a double helix, is stable, and stays in the nucleus.

RNA

A single-stranded molecule (with rare cases of double-stranded RNA) that can fold into complex 3D shapes, is relatively unstable, and can leave the nucleus.

ncRNA

Non-coding RNA that sometimes acts as molecular machines and is essential to life by facilitating cellular processes, protecting the cell from invading viruses, and altering other biomolecules.

mRNA

Messenger RNA that carries the genetic code from the nucleus to ribosomes for protein assembly.

tRNA

Transfer RNA that carries amino acids to the ribosome and translates the genetic code of mRNA into the amino acid sequence of a protein.

Ribosome

A protein-making machine composed of two subunits (small and large) made of ribosomal RNA and proteins. It links amino acids together to make proteins according to the mRNA sequence.

RNase

Ribonucleases, enzymes made of RNA, that break down other RNA molecules. They can get rid of unwanted RNA, refine RNA molecules by chopping off unwanted sections, and serve as a defense against RNA viruses.

Spliceosomes

Complexes of RNAs and proteins that remove introns and stick the remaining pieces back together in mRNA, allowing one mRNA to make many proteins through alternative splicing.

Self-Copying RNA

Hypothesized simple RNA molecule that can copy itself without assistance from other molecules, potentially playing a role in the origin of life on Earth.

pRNA

A component of the strongest nanomotor that packs genetic information into a virus's protein shell, requiring ATP for energy.

RNA interference (RNAi)

A defense mechanism against invading viruses observed in bacteria, plants, and some animals. It involves specialized RNAs and proteins that detect and chop up viral RNA, as well as RNAs and proteins that destroy viral mRNA.

Riboswitches

RNA structures that can switch from one shape to another when they detect specific molecules, changing their function in the cell.

G-C bonds

Strongest bonds between RNA bases, important for stabilizing RNA stacks and preventing loops from closing in on themselves.

A-U bonds

Bonds of medium strength between RNA bases, used for breaking bonds and placing G-C bonds in RNA molecules.

U-G bonds

Weakest bonds between RNA bases, used for breaking bonds and placing A-U bonds in RNA molecules.

RNA transcript

Product of RNA splicing in eukaryotic cells

RNA splicing

Processing step in the nucleus that removes introns from RNA transcripts

Structural RNA

RNA molecules that fold into three-dimensional structures with structural and catalytic roles in the cell

Regulatory RNA

RNA molecules that primarily act as regulators of gene expression

Exons

Segments of genes that are transcribed into RNA and eventually translated into protein

Introns

Segments of genes that are transcribed into RNA but are not translated into protein

Gene expression

The process by which a cell produces RNA from its genes, which can be controlled according to the cell's needs

Transcription

The process of copying a portion of DNA into an RNA sequence

Ribonucleotides

Nucleotides in RNA that contain ribose instead of deoxyribose and uracil instead of thymine

RNA polymerase

Enzyme that catalyzes the synthesis of RNA using a DNA template

Phosphodiester bonds

Bonds that link nucleotides together to form a linear chain in RNA

RNA chain growth

The process of elongating an RNA chain by adding nucleotides one at a time

RNA transcript release

The process by which RNA molecules produced by transcription are released from the DNA template

RNA transcripts length

RNA transcripts are much shorter than DNA molecules

Sigma factor

Subunit of bacterial RNA polymerase that assists in recognizing the starting point of transcription

Promoter

A specific sequence of nucleotides in DNA that indicates the starting point of transcription

RNA composition

RNA is a polymer made of adenine, cytosine, guanine, and uracil nucleic bases with a phosphate and ribose sugar backbone

RNA structure

RNA is single-stranded, allowing it to form more shapes via folding compared to double-stranded DNA

Base pairings

RNA forms strong (G:C), medium (A:U), and weak (G:U) base pairings

Unusual bases in RNA

Modified bases in RNA that stabilize RNA structure and enrich base pairing

Non-Watson-Creek base pairs

Base pairs in RNA that help stabilize RNA structure and form triple helix with DNA

RNA folding levels

RNA has primary, secondary, and tertiary structures that are essential for its functions

RNA secondary structure

The folding of RNA based on canonical base-pairing, which is easier to predict and more stable than tertiary structure

Assays for RNA secondary structure

Techniques such as RNAase digestion, DMS-seq, and SHAPE-seq used to study RNA secondary structure

RNA tertiary structure

The three-dimensional folding of RNA, stabilized by hydrogen bonds, stacking interactions, metal ions, and interactions between nucleotides

Multifactorial properties of RNA

RNA has various functions in the cell, including replication, gene expression, transcription, splicing, and more

key differences between RNA and DNA

RNA:

- ribose sugar (less stable)

- uracil (additional pairing: G-U)

- single-stranded (different shapes)

DNA:

- deoxyribose sugar

- thymine

- double stranded

unusual RNA bases Types:

- Pseudouridine (most common)

- Inosine

- Ribothymidine

- Dihydrouridine

unusual RNA bases Functions:

- post-transcriptional modification

- stabilize RNA

- modify base pairing

primary structure of RNA

sequence of nucleotides

secondary structure of RNA

canonical base pairing; more stable than tertiary structure; stable secondary structures minimize free energy

tertiary structure of RNA

3D structure (duplex, triplex, etc.); stabilized by H-bonds and stacking interactions

natural ribozymes

- peptide bond formation

- phosphodiester bond cleavage

- RNA ligation

artificial ribozymes

- RNA phosphorylation

- RNA aminoacylation

- glycosidic bond formation

aptamers vs ribozymes

- aptamers are small RNAs which bind specific ligands

- ribozymes are small RNAs which catalyze desired reactions

RNA Polymerase in Prokaryotes:

- one five-subunit enzyme

- sigma factors are used to guide RNA Pol to the promoter

RNA Polymerase in Eukaryotes:

- RNA pol I = rRNA (5.8S, 18S, 28S)

- RNA pol II = mRNA, lncRNA, snoRNA, miRNA, siRNA, most snRNA; structurally similar to bacterial RNA pol but has 12 subunits

- RNA pol III = tRNA, rRNA (5S), some snRNA

RNA pol vs DNA pol similarities

- synthesis is guided by a template

- synthesis is 5' -> 3'

RNA pol vs DNA pol differences

- rNTP (RNA Pol) dNTP (DNA Pol)

- no primer needed for RNA Pol

- only one strand synthesized in transcription

- transcription not limited to S-phase

- transcription is more error prone

prokaryotic transcription initiation

- RNA polymerase binds to the promoter sequences (-35 TTGACA and -10 TATAAT for sigma70)

- the closer the promoter is to the consensus sequences, the stronger the binding of RNA pol

- some genes have an upstream promoter which strengthen the binding; binds alpha CTD of RNA pol

- RNA pol forms a closed complex (DNA wound)

- open complex forms and DNA is unwound at the -10 sequence (pribnow box)

- Mg2+ dependent isomerization further unwinds the DNA

- many RNA pol can transcribe a gene at the same time

prokaryotic transcription elongation

- elongation is not smooth because RNA pol pauses due to RNA secondary structures, difficult sequences, backtracking, limited NTPs

- translation occurs co-transcriptionally

- avg elongation rate is 45 nt/s

prokaryotic transcription termination

- in Rho-independent, G-C rich inverted regions form a hairpin loop which destabilizes the 5' RNA-DNA hybrid and a poly-A DNA region (poly-U RNA region) leads to weak pairing and the collapse of the transcription bubble

- in Rho-dependent, Rho binds to the C-rich RUT site on the RNA, uses ATP to travel towards RNA pol, and when RNA pol stalls due to hairpin, Rho unwinds the hybrid

- very little post-transcriptional modification is done; bacterial mRNAs have a half-life of minutes; degraded by endonucleases (RNase E and RNase III) and exonucleases (RNase II and PNPase)

- polyadenylation in bacteria leads to degradation

eukaryotic pol II core promoters

for mRNA or miRNA

- TATA

for mRNA, piRNA, tiRNA

- CpG island

- ATG desert

eukaryotic pol II regulatory regions

- located several kbp upstream

- activator proteins bind to enhancer regions

- when DNA loops around the nucleosome, activators are connected to the basal transcription factors through the mediator

TFIID

- TBP subunit recognizes the TATA box

- TAF subunit recognizes other DNA sequences near the transcription start point; regulates DNA binding

TFIIB

- recognizes BRE element in promoters; accurately positions RNA pol at the start site of transcription

TFIIF

- stabilizes RNA pol interaction with TBP and TFIIB; helps attract TFIIE and TFIIH

TFIIE

- attracts and regulates TFIIH (helicase)

TFIIH

- unwinds the DNA at the transcription start point, phosphorylates Ser5 of the RNA pol CTD; releases RNA pol from the promoter

RNA pol II basal transcription factors

1. TFIID

2. TFIIB

3. TFIIF

4. TFIIE

5. TFIIH

eukaryotic transcription initiation

- TFIID (using TBP) binds to the TATA box; this induces a bend in the DNA

- TFIIB is recruited to the TBP-TATA box complex; this promotes recruitment of RNA pol with TBIIF to the promoter

- TFIIE and TFIIH join the complex; pre-initiation complex complete

- TFIIH unwinds dsDNA and phosphorylates the C-terminal domain of Rpb1 which stimulates the release of the mediator

- general transcription factors released

- transcription starts and elongation factors bind

*not the only way to begin transcription

eukaryotic transcription elongation

- nascent transcripts are co-transcriptionally modified (phosphorylations of Rbp1 CTD serines)

- phosphorylation of S5 leads to mRNA capping and recruitment of splicing factors

- phosphorylation of S2 leads to polyadenylation and recruitment of termination factors

- phosporylation of S7 is important for transcription of snRNAs

eukaryotic transcription termination

- Pol II continues passed the polyadenylation signal

- pre-mRNA is cleaved 11-30 nt after the beginning of the signal

- polyA polymerase adds a polyA tail which protects against degradation from 3' exonucleases

3 models for eukaryotic transcription termination

1. allosteric model - binding of 3' processing factors leads to rearrangement of elongation complex and termination

2. torpedo model - nuclease degrades nascent RNA from 5' end, catches up with the elongation complex, and displaces Pol II from DNA

3. combination model - a combination of 1 and 2

eukaryotic post-transcriptional modifications

7mG cap:

- added to the 5' end of all Pol II transcripts

- catalyzed by guanylyl transferase and methyl transferase

- held by a 5' -> 5' phosphodiester linkage

- helps with initiation of translation and protects the transcript from 5' exonucleases

polyadenylation:

- added to the 5' end of all Pol II transcripts (except snRNAs and mRNAs which code for histones)

- helps with initiation of translation and facilitates termination and splicing

splicing:

- major spliceosome: U1, U2, U,4 U5, U6 subunits; intron boundaries GU -> AG

- minor spliceosome: U11, U12, U4atac, U5, U6atac; intron boundaries AU -> AC

- splice sites recognized by base pairing w/snRNAs

- catalysis performed by U6 RNA (ribozyme)

nuclear pre-mRNA

- intron class

- used for most eukaryotic genes

- transesterification (A branch site)

- major and minor spliceosomes

group II introns

- some organelle genes and prokaryotic genes

- transesterification (A branch site)

- self-splicing ribozyme

group I introns

- rRNA, organelles, some prokaryotes

- transesterification (G branch site)

- self-splicing ribozyme

tRNA introns

- tRNA only

- endonuclease, ligase

RNA base editing:

- most common in tRNA but present in rRNA and mRNA

- A-to-I editing (ADARs, synaptic transmission, miRNA processing); important for neuronal development and innate immunity

- C-to-U editing (apolipoprotein, HIV)

mRNA methylation:

- transient (reversible)

- m6A is the most common

- readers (recognize/bind modified bases), writers (modify the base), erasers (reverse modification)

- increases/decreases stability, increases/decreases translation, modulates splicing

rRNA transcription

28S, 18S, and 5.8S rRNAs (transcribed by RNA pol I) are processed from a single transcript

tRNA transcription

tRNAs (transcribed by RNA pol III) are processed by the ribozyme RNase P

mRNA quality control

degradation can occur by RNases, exonucleases, or endonucleases

mRNA quality control non-stop decay (NSD):

detects a transcript without a stop codon and degrades it 3' -> 5'

mRNA quality control no-go decay (NGD):

detects a transcript without a start codon and cuts it into 2 fragments to degrade it both ways

mRNA quality control nonsense mediated decay (NMD):

detects a transcript with a premature stop codon and cuts it into fragments

regulation of gene expression by miRNAs and siRNAs

- RNA-induced silencing complex (RISC)

- functions to suppress translation by binding to the 3' UTR

- imperfect pairing = suppression of translation

- perfect pairing = degradation of transcript

different locations from where lncRNAs are transcribed

- intergenic: no overlap with other genes

- intronic: in the intron of another gene

- sense: overlap with another gene, same orientation

- antisense: overlap with another gene, antisense orientation

- bidirectional: head to head with another gene

lncRNA functions

- regulation of chromatin structure (cis and trans silencing)

- regulation of gene expression by lncRNA transcription (activation or silencing)

- regulation of transcription in cis or trans (activation/repression of transcription factors/accessory proteins; TF recruitment or sequestration)

- post transcriptional regulation (binding of antisense transcripts; gene silencing pathways)

- most function in the nucleus; some function in the cytosol

regulation of gene expression in bacteria can be:

- constitutive (expressed all the time)

- repressible (normally on but can be turned off by a repressor)

- inducible (normally off but can be turned on by an inducer)

lac operon +/- lac/glu

when lactose is not available:

- lac repressor protein is transcribed by lacI

- lac repressor protein binds to the lac operator so transcription is inhibited

- RNA pol cannot bind to the promoter

when lactose is available:

- lac repressor protein is transcribed by lacI

- lac repressor protein is bound by allolactose so it cannot bind to the operator

- RNA pol can bind to the promoter so transcription can proceed

when glucose is unavailable + lactose is avail:

- cAMP levels rise

- cAMP binds to CAP and the complex binds to the CAP-binding site upstream of the promoter

- this complex helps RNA pol strongly bind to the promoter (high levels of transcription)

when glucose is avail + lactose is avail:

- cAMP levels fall

- CAP cannot bind to the CAP-binding site

- RNA pol weakly binds to the promoter (low levels of transcription)

Trp operon REPRESSION when Trp is low

- Trp repressor protein is made but cannot bind to the operator

- RNA pol binds to the promoter and transcription can proceed

Trp operon REPRESSION when Trp is high

- Trp repressor protein is made

- Trp binds to the Trp repressor and the complex binds to the operator

- RNA pol cannot bind to the promoter and transcription stops

Trp operon ATTENTUATION when Trp is low

- Trp-tRNA is low

- ribosome stalls at Trp codons

- region 2 binds region 3 (alternate loop) and transcription continues (Trp genes transcribed)

Trp operon ATTENTUATION when Trp is high

- Trp-tRNA is high

- ribosome doesn't stall at the Trp codons

- region 3 binds region 4 (terminator hairpin) and transcription ends (Trp genes not transcribed)

other prokaryotic gene regulation mechanisms

- riboswitches (changes in secondary structure)

- small RNA (complementary base pairing w/mRNA

chromatin modification

- heterochromatin restricts gene expression

- histone modification (acetylation=increased transcription; methylation=decreased transcription)

transcriptional regulation

- different Pol II promoter sequences

- regulatory elements are spread out

- transcription factors bind specific sequences

- transcription factors bind cooperatively (one recruits more)

- one regulator can regulate many genes

- one regulator can have different effects depending on cell type, gene, etc

- reprogram differentiated cells

post-transcriptional regulation

- alternative splicing

- miRNA suppression or degradation

- lncRNA transcription and binding to DNA to recruit activators and repressors

regulation of gene expression in eukaryotes

1. chromatin modification

2. transcriptional regulation

3. post-transcriptional regulation

example:

- RXR nuclear receptor; retinoid X receptors which are ligand-activated transcription factors which function to recruit coregulators

eukaryotic signal recognition particle is composed of:

proteins and 7SL RNA

4.5SL RNA

prokaryotic signal recognition particle

about RNA pol II

- it is composed of 12 subunits

- promoters can be bidirectional

- heteochromatin is not more accessible to it

about lncRNAs...

all lncRNAs are transcribed from introns of coding genes