Carboxy terminal domain, business end, the thing that polymerizes the dna
In all species, you have these repeats of amino acid sequence, arent perfect repeats, increase complexity of euk, the number of repeats increases (yeast-26, humans-52) increase length and number
-will notice that amino acids 2,5,7 are serine, very important in putting and taking off accelerator in rna pol2

The preinitiation complex forms with the hypophosphorylated form of RNA polymerase II Then TFIIH phosphorylates serines 2 and 5 in the heptad repeat in the carboxyl-terminal domain (CTD) of the largest RNA polymerase subunit creates the phosphorylated form of the polymerase enzyme (IIO) This phosphorylation is essential for initiation of transcription

Elongation complex continues elongating the RNA when: Polymerase CTD is further phosphorylated by TEFb NTPs are continuously available TBP, and TFIIB remain at the promoter TFIIE and TFIIH is not needed for elongation and dissociate from the elongation complex.

CDK9 is a cyclin-dependent kinase and it is a component of the multiprotein complex TEFb, which is a elongation factor for RNA polymerase II.

TFIID lands on promoter at the TATA, TFII A and B then add, This is followed by the RNA pol II with attached TFII F, then TF II E and H attach, Finally an ATP is burnt and the trascirption can begin as the Pol is phosphorylated

Consensus sequences are part of the promoter regions that ensure transcription factors bind (DNA bnding proteins direct RNA polymerase to the right location) Ex. CAAT, TATA, TATAAT

Binding of RNA pol II tp promoters requires that TFIIA and TFIID initially to the TATA box of the core promoter, TFIIB then binds directly to TFIID, then RNA polymerase binds to the complex, and DNA is aligned to transcription start point, TFIIE, F, and H, bind and an ATP and Go

rRNA, nucleous. rRNA genes are located in the specific chromosomal region tered nucleolar organizer

(tRNA synthesis) RNA polymerase III recognizes a split promoter located in the 5' region flanking the coding region. precursor is 100 nucelotides long, and assumes clover leaf shape, subsequently cleaved by endonucleases at both ends, 3' terminal gets a CCA-OH to connect to amino acid

= TBP + TAFs -TAFs (TBP Associated Factors) with TBP and RNA pol recognize INR -partially unwinds DNA/binds TATA box

TFIIB binds BRE promoter sequence, positioning RNA core polymerase like sigma factor would

-triggers recruitment of RNA processing enzymes (5' cap, splicing, poly(A) tail) -when processing complete a different serine is phosphorylated, and elongation continues

RNA polymerase II (RNAP II and Pol II) is a multiprotein complex that transcribes DNA into precursors of messenger RNA (mRNA) and most small nuclear RNA (snRNA) and microRNA

CpG islands are short streches of DNA with an unusually high GC content and a higher frequency pf CpG dinucleotides.They are located in the 5' regulatory regions of genes. They are associated with all housekeeping genes and up to 40% of tissue specific.
!Methylation of a CpG island prevents activation of a promoter within it

Commitment complex (not yet any chemical activity) U1 base paired with 5' splice site

U2 addition to base pair with branch site (ATP required)

Joining of U4/6 and U5 tri-snRNPs

U1 and U4 release. U6 base pairs with the 5' splice site
U6 also base pairs with U2
U2 remains base paired with branch site
U5 intreacts with both exons though its loop

is formed by adding a G to the first base of the transcript via 5'->5' link by guanine 7-methyltransferase.The capping process takes place co-transcriptionally. is important for splicing, transport and translation initiation

is a complex which is used for removing introns in eukaryotes.They contain snRNAs.

1⃣ The sequence AAUAAA is a signal for cleavage to generate a 3′ end of mRNA that is polyadenylated.

2⃣ The specificity factor and endonuclease cleave RNA downstream of AAUAAA.

3⃣ The specificity factor and poly(A) polymerase add about 200 A residues processively to the 3′ end.

The poly(A) tail controls mRNA stability and influences translation.

For Group 1 introns, there is a G-factor for splicing (No protein involves here)
For Group 2, Lariat structure forms(No protein involves in vitro)

During S phase need for histones is increasing
Expression is high in S phase

RNA pol III terminates at poly(U)4 sequence embeded in a GC-rich sequence

Does not mediated by spliceosomes, instead by successive cleavage and ligation reaction

Nuclease ➡ Folding ➡ Ligase

RNA polymerase I terminates transcription at an 18-base terminator sequence.
The large and small rRNAs are released by cleavage from a common precursor rRNA
the 5S rRNA is separately transcribed

(by endonucleases,exonucleases)

The C/D group of snoRNAs is required for modifying the 2′ position of ribose with a methyl group.
Methylation of rRNAs and/or tRNAs has crucial functional roles in structural stabilization, codon-anticodon interactions, and wobble base pairing

The h/ACA group of snoRNAs is required for converting uridine to pseudouridine.

In each case the snoRNA base pairs with a sequence of rRNA that contains the target base to generate a typical structure that is the substrate for modification.

H/ACA snoRNAs have two short, conserved sequences and two hairpin structures, each of which has regions in the stem that are complementary to rRNA. Pseudouridine is formed by converting an unpaired uridine within the complementary region of the rRNA.

1. Removal of terminal Pi (recall that there are 3 P) from 5' end of nascent (newly synthesized) RNA transcript
2. Guanine cap is attached to 5' end using GTP (GTP loses 2 phosphates)-> this is a 5'to5' linkage!!
3. Methylation of guanine and some ribose the cap is methylated guanosine (has only one phosphate)

All cellular mRNAs are eventually degraded.

mRNA decay is a highly regulated process controlling gene expression at the post-transcriptional level, often concomitantly with regulation at the transcriptional level

Half-life is controlled by specific interactions between cis-acting mRNA elements and the proteins that bind these elements.

Changes in mRNA decay allow for fine-tuning of gene expression and can occur in response to developmental or environmental stimuli such as nutrients, cytokines, hormones, and temperature shifts, as well as environmental stresses such as viral infection, hypoxia, or tissue injury.

The time it takes to degrade half of existing mRNA molecules.

Eukaryotic mRNA half-lives can vary from minutes to hours.

To determine the half-life drugs used which shutdown RNA pol II

DCP2-DCP1
mRNA decapping enzyme

Scavenger decapping enzyme decaps removal parts from the 3' to 2' exonucleolytic pathway

Exosome
Consists of three heterodimers.It is evolutionary conserved.

General mRNA degradation
Ski2 helicase is required for activity

ARE mediated decay
Destabilizing element (DE) binding proteins can recruit deadenylase and then exosome

Nonstop decay
There is no termination codon. This is toxic.
Recruit the exosome by Ski7.

Rrp44 also has endonucleolytic activity

Endonuleolytic cleavage (PMR1, RNase, MRP, IRE1, SMG6)

CAF1-CCR4-NOT1
Inhibited by PAB
No effect of 5' cap structure

PAN2-PAN3
Stimulated by PAB
Effect of 5' cap structure unknown
Initially work on newly synthesized polyA and shorten them to a point where mRNA is stable

PARN
Inhibited by PAB
Stimulated by 5' cap structure
Controls maternal mRNAs

Lsm1-7 is a poly(A) binding protein and required for recruitment of Dcp

As a general rule, mRNA- spesific determinants of mRNA stability reside within the 3'-UTR of the mRNA

They ofthen target the mRNA decay from the 3' end.
AU rich elements (AREs) are common destabilizing elements

They often protect the mRNA from endonucleolytic clevage
Occur highly stable mRNAs

Prokaryotic mRNAs

Most of the mRNAs are polycistronic(have more than one coding region).
Generally have stem-loop structure (for termination of transcription.)

Degradation is quite rapid and it's initiated 5' triphosphate and yield monophosphate. Then exo and endonucleolytic cleavage can start.

3' polyadenylation can facilitate the degradation of mRNA fragments containing secondary structure

poly(A) polymerase (PAP) adds the stretch of polyadenylic acid to the 3' end of the eukaryotic mRNA.

PNPase is 3' to 5' exonuclease

Eukaryotic mRNAs
Have capping region, very long Poly(A) tail, and also PABP.
We may see secondary structures.
Histone mRNAs do not have poly(A) tail but have stem-loop structure.

mRNA stabilized by IRE-binding protein. IRE stays in mRNA and inhibit degretarion mechanism phsically. If Fe levels high, IRE can not bind mRNA anymore and mRNA becomes unstable

Abnormel nuclear RNAs are identified and destroyed by RNA surveillance system.

The yeast TRAMP comlex recruits the exosome to abnormal RNAs and facilitates its 3' to 5' exonuclease activity.

A protein required to terminate polypeptide translation to cause release of the completed polypeptide chain and the ribosome from mRNA.

Exon junction complexes (EJC) recruits Upf1 and SMGs that will lead to degradation

SKI protein force and stimulate release of the ribosome and mediates decay.

Ribosome may stall while translation event. This may happen because of the secondary structure of mRNA that can not be resolved, or rare tRNAs in the cell. (rare codons)

Formed by aggregation of translationally silenced mRNA and many different proteins.

Germ cell granules and neuronal granules function in translational repression and transport.

Cytoplasmic ribonucleoprotein (RNP) granules primarily composed of translationally repressed mRNAs and proteins related to mRNA decay, suggesting roles in post-transcriptional regulation. P-bodies are conserved in eukaryotic cells and exhibit properties of liquid droplets.

Accumulate in response to stress-induced inhibition of translation. Certain mRNA species can be stored.

Three main functions of mRNA localization

1⃣ Pattern formation and fate spesification in oocytes and embryos.

2⃣ Generation of different daughter cells in asymmetric cell division.

3⃣ Compartmentalization of a cell into specialized regions.

Ash1 mRNA example from budding yeast?

like a guarantee mechanism to make sure that mutated mRNAs are not translated.

If there is a stop codon mutated upstream of actual stop codon, ribosome cannot disrupt EJC and decay begins.

is going to recruit nucleases to initiate degredation of the RNA.It binds to EJC downstream of a stop codon

It accompanies mRNAs from the nucleus to the cytoplasm, where it is removed by the first round of translation, and recycled back into the nucleus.

due to destabilization and/or a conformational change of the Pol-ll after transcribing poly-A site. Pol II becomes less processive ant it will fall of

due to endonucleolytic cleavage at poly-A site for the 5'->3' exonuclease. Remaining RNA molecules become target to exonucleases and such enzymes degrate RNA more faster than pol II is transcribing.

General Information
Rare examples of bacteria but more common in unicellular or oligocellular eukaryotes
They do not require any other protein function for this process to happen.
In Tetrahymena, group I introns are found in genes for rRNA genes
Splicing occurs by two transesterification reactions, without requiring energy input.

Process
1⃣ The 3′–OH end of the guanosine cofactor attacks the 5′ end of the intron in the first transesterification.
2⃣ The 3′–OH end generated at the end of the first exon attacks the junction between the intron and second exon in the second transesterification.
3⃣ The intron is released as a linear molecule that circularizes when its 3′–OH terminus attacks a bond at one of two internal positions.

Group I introns form a secondary structure with nine duplex regions.

The cores of regions P3, P4, P6, and P7 have catalytic activity.

Regions P4 and P7 are both formed by pairing between conserved consensus sequences.

A sequence adjacent to P7 base pairs with the sequence that contains the reactive G.

Ribozymes (ribonucleic acid enzymes) are RNA molecules that have the ability to catalyze specific biochemical reactions, including RNA splicing in gene expression, similar to the action of protein enzymes.

example: glms gene in Gram positive bacteria
Responsive to glucosamine-6-phosphate levels

Some introns have ORF that encodes for an endonuclease that breaks double-stranded target DNA (homing endonuclease)

The intron transposes into the site of the double-strand break (by a DNA mediated replicative mechanism

Group I introns are large self-splicing ribozymes. They catalyze their own excision from mRNA, tRNA and rRNA precursors in a wide range of organisms.

Group II introns are a large class of self-catalytic ribozymes and mobile genetic elements found within the genes.

Ribozyme activity can occur under high-salt conditions in vitro. However, assistance from proteins is required for in vivo splicing.

In contrast to the group I introns, intron excision occurs in the absence of GTP and involves forming a lariat, with an A-residue branchpoint strongly resembling that found in lariats formed during splicing of nuclear pre-mRNA.

A single reading frame specifies a protein with reverse transcriptase maturase, DNA-binging motif, and a DNA endonuclease activity

maturase activities assist folding into the active catalytic structure. (for autosplicing)

Viroids are infectious, no protein coat.
Virusoids are encapsidated by a plant virus.

Form a hammerhead structure that has a self cleaving activity.

Example: Apolipoprotein-B and glutamate receptor mRNAs have site-specific deamination catalyzed by cytidine and adenosine deaminases that change the coding sequence.

CAA codon at a specific place changed to UAA by deamination. Since UAA is the stop codon, edited RNA translates shorter protein.

Extensive RNA editing in trypanosome mitochondria occurs by insertion or deletions of uridine.

The mRNA editing in the trypanosome cosII gene lead to frameshift since uridine addition

The guide RNA provides the template for addition (or less often, deletion) of uridines.

Editing is catalyzed by the editosome, a complex of endonuclease, exonuclease, terminal uridyl transferase activity, and RNA ligase

An intein has the ability to catalyze its own removal from a protein in such a way that the flanking exteins are connected

Protein splicing is catalyzed by the intein.

Most inteins have two independent activities: protein splicing and a homing endonuclease.

Chapter 21

Chapter 19

Chapter 18

The effect of splicing enhancers and silencers is mediated by sequence-specific RNA binding proteins, many of which may be developmentally regulated and/or expressed in a tissue-specific manner.
The rate of transcription can directly affect the outcome of alternative splicing.

Trans-splicing is a special form of RNA processing where exons from two different primary RNA transcripts are joined end to end and ligated.
(SL RNA)

RNA whose activity is controlled by small ligand, which may be a metabolite product.

Riboswitch may be ribozyme, which means binding a substance will lead to formation change and some activities for that RNA. Increasing a byproduct in the cell can lead to activation of riboswitch and clevage of the RNA stops activation. It is a kind of translation contol mechanism.

piRNA transcribed in clustered areas. It is expressed actively in the germline. Precursor is prossesed to piRNA intermediates. piRNA loaded with Piwi protein (Yb body) and after 3' end trimmig and 2'-O methylations happens, Piwi-piRNA complex gets into nucleus. Purpose of this complex is to prevent transposable elements activity.

A regulator RNA can function by forming a duplex region with a target RNA that may block initiation of translation, cause termination of transcription, or create a target for an endonuclease.

Transcriptional interference (TI) occurs when an overlapping transcript on the same or opposite strand prevents transcription of another gene.

Long ncRNAs (lincRNAs) are defined as longer than 200 nucleotides, without an open reading frame, and produced by RNA Pol II.

A gene that codes for an (antisense) RNA that has a complementary sequence to an RNA that is its target.

Vast tracts of the eukaryotic genome are transcribed on both strands.

Functions

✅ Interact with promoter element.
✅ Recruitment of chromatin factors, interactions with transcription factors
✅ Acts as a decoy for a transcription factor.
✅ Modulate splicing/translation
✅ Act like sponges and recruit miRNA.

Importance

Correlate with an increased ability of multicellular organisms to differentiate into many different cell types.
This allows an organism to achieve greater diversity from the same number of protein-coding genes.

Lower organisms: mRNA>lncRNA
Higher organisms: lncRNA >> mRNA

Example
XIST, nuclear compartmentalization, and propagation of H3K27 methylation, X chromosome inactivation.

Small Cajal body-specific RNAs (scaRNAs) are a class of small nucleolar RNAs (snoRNAs) that specifically localise to the Cajal body, a nuclear organelle (cellular sub-organelle) involved in the biogenesis of small nuclear ribonucleoproteins (snRNPs or snurps).

Repeat associated small interfering RNA (rasiRNA) is a class of small RNA that is involved in the RNA interference (RNAi) pathway. RasiRNA are in fact Piwi-interacting RNAs, which are small RNA molecules that interact with Piwi proteins

RNA that has a complementary sequence to an RNA that is its target.

Antisense RNA can be generated by reversing the orientation of a gene with respect to its promoter.

Main fucntion is to match with RNA and deactivate it.

microRNAs are small RNA particles that are transcribed by cells to downregulate the gene expression. They attach with the complementary sequence in RNA 3' UTR and targets for deadenylation, degradation or cleavage, and translational repress. Parts of the mRNA sequences that miRNA binds are highly conserved.

It interferes with the expression of specific genes with complementary nucleotide sequences by degrading mRNA after transcription, preventing translation.
Small siRNA is synthesized by DICER, it needs dsRNA or shRNA. siRNA can bind anywhere in the mRNA and is degraded.

endo-siRNAs synthesized from repetetive regions. Instead of providing gene silencing, they recruit heterocromatin modifiers to keep that regions more heterocromatic.

The self-splicing of group I introns occurs in a series of transesterifications that require no hydrolysis for energy

TTP: thiamine pyrophosphate is a vitamin B metabolite. When TPP binds riboswitch, riboswitch goes under a structural change and consequently processed mRNA will not be translated.

Requires a factor and it binds to downstream of termination site.

Transcribes a terminator sequence to produce a terminator sequence to produce a region rich in uracil residues on the transcript

Recognizes the AAUAAA sequence and cleaves the RNA for release.

Chapter 29