0 Posted 2021-11-27Updated 2024-01-11Notes / Class / Tulane / Biochemistry29 minutes read (About 4391 words)

Catalytic RNAs: Splicing and Translation

Posttranscriptional Modification of Eukaryotic RNAs

Role of the 5’ cap

Ribosomal recognition during translation

Cap structure

7-methylguanosine (m7G)
Joined to the mRNA first nucleotide
Via a 5’-5’ tri-P bridge

Involved enzymes

RNA triphosphatase removing the γ-P of the mRNA’s 5’ site
mRNA guanylyltransferase (a capping enzyme) adding GMP
Guanine-7-methyltransferase (a capping enzyme) methylating guanine
Capping enzymes bind to RNAP-II, which will switch RNA synthesis initiation to elongation

Poly(A) tails

~250 nt (~80 nt in yeast)
Cleavage and polyadenylation specificity factor (CPSF), cleaving up to 35 nt past the AAUAAA sequence
Poly(A) polymerase generate poly(A) tail using ATP.
CPSF binds to RNAP-II, coupling polyadenylation to transcription termination.
Poly(A) tail binds to poly(A)-binding protein, protecting from degradation, increasing mRNA stability.

Exons and Introns

Precursor mRNAs (pre-mRNAs) are processed by the excision
of introns and the splicing (joining) of exons

Exon Splicing in Two-Stage Reactions


© Zhichao Tang, 2021

Invariant sequences for splice junction:

GU at the intron’s 5’
AG at the intron’s 3’
A branch point (Intron A) near the 3’ splice site
Free guanosine, GMP, GDP, or GTP not part of the intron

A 2’-5’ P-diester bond between the intron A (OH2’) and the intron at the 5’ splice site, forming a “lariat” structure.
The Exon 1 OH3’ group at the 5’ splice site form a 3’-5’ P- diester bond with the Exon 2 at the 3’ splice site, releasing the intron with the free OH3’ group.
The intron keeps the lariat structure. Note: Splicing proceeds w/o free energy lose. Cleavage of one P-diester bond and formation of a new bond.

Note: Splicing proceeds w/o free energy lose. Cleavage of one P-diester bond and formation of a new bond.

Spliceosome-aided RNA splicing


60S spliceosome particle containing five small nuclear RNAs + ribonucleoproteins (U1, U2, U4, U5 and U6) 1. U1 recognizes the 5’ splice junction 2. U2 recognizes the branch point (intron A) 3. The binding of U4-U5-U6 forms spliceosome. 4. RNA cleavage at the 5’ splice site 5. RNA cleavage at the 3’ splice site 6. Intron degregradation	© utexas.edu

Self-Splicing RNA


© Chen Zhao, 2017

Group II intron

A bulged adenosine in Domain 6 attacks the 5’ splice site, forming a lariat intermediate (a 2’-5’ linkage)
The 5’ and 3’ exons are ligated and the lariat intron is released


© Chang HoLee, 2018

Group I intron: In the presence of guanosine, GMP, GDP, or GTP, the pre-rRNA is self- splicing.

The 3’-OH of guanosine attacks the 5’ splice site, forming a P-diester bond with the intron.
The 3’-OH of the exon 1’s 5’ end attacks the 3’ splice site of exon 2, joining the two exons.
The 3’-OH of the intron attacks the nucleotide 15 residues from the intron’s 5’ end, yielding a cyclic form.

Summary of Spliceosome- aided Splicing and Self-Splicing

In group I introns, a guanosine cofactor (G) that is not part of the RNA chain associates with the active site. The 3’-hydroxyl group of this G attacks the 5’ splice site;

the reaction is similar to those involving the 2’ hydroxyl groups of branch sites as in group II introns and RNA introns spliced in spliceosomes.

The subsequent trans-esterification that links the 5’ and 3’ exons is similar in all three splicing mechanisms.

Interactions of RNAP-II with Capping enzyme, splicing and CPSF

Aminoacyl-tRNA Synthetases


© Bioforums

Attach amino acids to tRNAs
Two step reactions
- Amino acid activation by the reaction with ATP
- Formation of an aminoacyl-tRNA
The synthetase enzymes have an elongated shape.
Binding the anticodon of tRNA near the one end of the enzyme.
Binding the a.a. acceptor stem of tRNA near the other end.

The bases of the anticodon are unstacked and splay outward, binding in separate recognition pockets of the Synthetase.

Proofreading by Aminoacyl-tRNA Synthetase (tRNA^thr Synthetase)


© Justin C. Morse

Binding to the amino acid substrate pocket of tRNA^thr Synthetase that consists of the zinc ion

Threonine (attached at the 3’ of tRNA) binds at this pocket b/c it can coordinate Zn²⁺ using its NH₂ and OH groups
Serine binds b/c it can coordinate Zn²⁺ using its NH₂ and OH groups!!
But valine cannot bind b/c it lacks the OH group.
tRNA^thr is tRNA for threonine.
- Attaching Thr to tRNA^thr makes Thr-tRNA^thr (tRNA is correctly charged)
- Attaching Ser to tRNAthr makes Ser-tRNA^thr (tRNA is mischarged!)
- Ser-tRNA^thr incubates with Thr-tRNA synthetase------>
- Rapid breakdown: Ser and free tRNA^thr !!
- Thr- tRNA^thr incubates with Thr-tRNA synthetase------>
  - No breakdown!
The flexible aa acceptor stem of tRNAthr can move the attached serine residue between the activation/catalytic site and the editing site.
Since serine fits well at the editing site, the residue is removed from tRNAthr .
Threonine is too large to fit at the editing site, the residue remains on tRNAthr .

Ribosomes

Prokaryotic 70S	Eukaryotic (80S)
30S subunit: 16S rRNA, 21 proteins ( bond mRNA (red))	40S subunit: 18S rRNA, 33 proteins
50S subunit: 5S and 23S rRNAs and 31 proteins [bound to three sites: exit (E), peptidyl §, and aminoacyl (A)]	60S subunit: 5S, 5.8S and 28S rRNAs and 49 proteins

Summary of the Composition and Mass of Ribosomes in Bacteria and Eukaryotes

Translation (Protein biosynthesis)

Translation (Protein biosynthesis) Protein synthesis proceeds from N-term to C-term by a peptidyl transferase activity
Chain elongation occurs by linking the growing peptide to the incoming tRNA’s aa.
The growing peptide is transferred from the P site to the incoming aa-tRNA in the A site, resulting in the formation of a peptidyl-tRNA with one more aa residue in the A site.
The peptidyl-tRNA is translocated to the P site to have an empty A site for a new aa-tRNA.
The uncharged tRNA in the P site moves to the E site (not shown) for
exiting.


© 1970 American Association for the Advancement of Science. Miller, O. L. et al. Visualization of bacterial genes in action. Science 169, 392–395 (1970).

Ribosomes read mRNA in the 5’ → 3’ direction.
Active translation occurs on polysomes

Multiple ribosomes can bind to a single mRNA transcript
Initiation
- Initiation codon(AUG) in mRNA, N-formylmethionyl-tRNA, 30S rRNA, 50S rRNA, Initiation factors (IF-1, IF-2, IF-3) GTP, Mg
Elongation
- Functional 70S ribosome (initiation complex), aminoacyl-tRNAs specified by codons, elongation factors (EF-Tu, EF-Ts, EF-G), GTP, Mg
- Peptidyl transferase activity not by rProtein, but by rRNA
Termination and release
- Termination codon in mRNA, polypeptide release factors (RF1, RF2, RF3), GTP

Translation - Initiation

Almost half of E. coli proteins begin with the uncommon N-formylated Met (fMet).
The fMet has an amide bond, which can grow only to the C-term side.
fMet is removed by deformylation, followed by the removal of resulting Met sometimes.

Selectivity for the Translation Initiation Site

In E. coli, base pairing interactions of an mRNA Shine-Dalgarno sequence with the 16S rRNA allows ribosome to select the proper initiation codon.

Selectivity for the Translation Initiation Site

Prokaryotic Translation - Initiation


© Gaurab Karki, 2017

IF-3 binds to the 30S subunit to dissociate the inactive 70S ribosome into 30S & 50S
mRNA and IF-2 forms a complex with GTP and fMet- tRNAfmet along with IF-1 bind to the 30S subunit.
IF-1 and IF-3 are released and the 50S subunit binds to the complex, stimulating GTP hydrolysis by IF-2. It is followed by IF-2 release.

“fMet-tRNA bound at the P site of 50S in the 70S initiation complex.”

Prokaryotic Translation - Elongation


© Gaurab Karki, 2017

Decoding: Ribosome binds an aa-tRNA, whose anticodon is complementary to the mRNA codon in the A site.
Peptide bond formation: The peptidyl group in the P site is transferred to the aminoacyl group in the A site
Translocation: EF-GGTP binding to A translocates tRNAs from “P and A” to “E and P”, releasing the free tRNA at E.
GTP hydrolysis releases EF-GGDP

Prokaryotic Translation – Termination


© Gaurab Karki, 2017

In E. coli, the stop codons (UAA, UGA, UAG) have no
corresponding tRNAs, recognized by release factors.

RF-1 recognizes UAA and UAG.
RF-2 recognizes UAA and UGA.

RF-1 (or RF-2) recognize corresponding
codons at the A site.
(1) The peptidyl group of tRNA at the P site is transferred to water, releasing the polypeptide.
(2) RF-3GTP binds to ribosome.
GTP hydrolysis releases RF-1 (or RF-2).
RF-3GDP is replaced by ribosome recycling factor (RRF), followed by EF-GGTP
Upon GTP hydrolysis, RRF, EF-GGDP, mRNA and tRNA at the P site dissociate, yielding an inactive 70S ribosome.

Inhibitors of DNA synthesis and their specificities

AZT and DDI: reverse transcriptase, chain terminate

nuceoside analogues of guanosine: phosphorylated by a viral kinase

Polymerase processivity Enhanced by a Clamp

β clamp in bacteria
PCNA in eukayotes
loaded onto dsDNA on calmp

Clamp loader couples ATP Hydrolysis to Clamp Loading

Sliding clamp + ATP: open and bind DNA; ATP → ADP, DNA releases

Biological advantages of DNA

Provides stable, yet mutable storage of genetic information. Metabolic stability and availability of repair mechanisms ensure long-term survival under a variety of physiological conditions.
Serves as a template for accurate and adaptable replication of genomes. This biological role ensures the transmission of important physiological functions for multiple generations.
Serves as a template for the expression and regulation of genetic information. This biological role facilitates adaptation of genomes to changing physiological environments.
Amenable to diversification (i.e., can evolve). Mutation and genetic recombination (DNA rearrangements) are the two major driving forces in the evolution of genomic diversity and adaptation in nature.

DNA ligases

Eukaryotic DAN ligase use ATP rather than NADP

initiation od DNA replication in E. coli

origin region (OriC) ~245bp

Duplex unwinding by DNA helicase

Exposed ssDNA allows SSB and helic …

Initiation: OriC-DnaA intercation → Entry of SSB and DnaB (hexlicase) → ENtry of promase → RNA primers are synthesized and the elongation complexes for 2 DNA replication forks are assembled

Elongation:

Prokaryotic DNA replication

A typical DNA replicsome

synthesis of leading strand are coupled with lagging strand by clamploader complex.

The e.coli Pol III holoenzyme …

Pols enyzme subunit factors

Eukaryotic DNA replication:

Pro: no cell cycle and start every time

Eu: onece anc only once in S phase; highly regulated

G1: Preparing
Checkpoint: end of the S phase

Time is variatd

Eukaryotic chromaomse have multiple origins…

Pro: single initial site
Euk: multiple initial site

Eukaryotic replication initiation is hihgly oreder… well regulated and complex…

Helicase (Mcm2-7)
Sld2/3 phosphorylated by CDK

Super Current Biochemical Reconstitution of Regulated replication origin firing

Termination in prokaryotes

Ter sites, 2 converging new ds circular DNA molecules separated from each other by recombination and with assistance of

Cell are constantly exposed to agents that can cause damage to their genome

Repair, tolerant, destroy the cell

Types of mutation

Transition: AT → G/C
Transversion: A/T → C/G
Frameshift: GGGGGG → GGGGG

Base alteration

Oxidation:
8-oxodG(antl)-dC(antf)
8-oxodG(synl)-dA(antf)

The rotation of the bound which cause the shift of the side chain and attribute difference hydrogen bonds. 3 to 2, C to A

Alkylation; Cross-links

Exogenous Damage: Exposure to ultraviolet radiation

Absorbed by adjacent pyrimidine bind and form double bond and cause mutation or lethal during replication/transcription

Repair

Base Excision Repair

Single Nucleotide

Nucleotide Excision Repair

oligonucleotide

Mismatch Excision Repair

mismatched base

Base Excision Repair

how recognize
AP site
Donw string/ leading string doesn’t matter
ligates involved

Nucleotide excission repair (NER)

recognize Enzymes (XPC)
recruit other factors
unwinding, mutation is exposed
Some protein was recruited and mistake was cut / rescued.

Recognition → resynthesis

Xeroderma pigmentosum (XP)

Accelerated skin cancer; mortality likely due to metastatic melanoma or squamous cell carcinoma

MMR pathway - E. Coli

MutS, MutL, MutH, ATP
MutH: cut the strand (down/leading)

methelation is critical here

RNA polymerases

Transcription using DNA to RAN
- Bacterial: ON enzyme for all of RNA synthesis
- Eukaryotic: At leased Three I II III
Strangs 1:
- Template;
- Non-coding;
- Anti-sense:
- Completed strand
Strands 2:
- Sense strand: coding strange of the DNA
- Non-completed strand
Bacterial promoter consensus sequences
- UP Element; -35; -10; RNA stra +1
- -35; -10: promoters (similar to regulatories in Eukaryotic.)

Expression controlled by interactions of promoter elements with RNA polymerase and specific repressor/Activators

Bacterial RNAP

Holoenyme versis core enzyme
Core subunits: α₂ββ’ω (400kDa)
Holoenzyme: core subunits plus factors
…

Five σ factirs in E. coli gene expression

Exp: (not important)
- σ 70: Most genes
- σ 32: Heat schock proteins
Trascription Elongation
- RANP trancks along the DNA template, synthesizes mRNA in te 5’ to 3’ direction and unwinds and rewinds the DNA as it reads.
- Transcription elongation causing DNA supercoiling, relaxed by topisomerases
Termination
- Transcription Terminattion
  - A series of 4 - 10 base
  - Sequence depended: CGGCGCTTTTTT (CG rich region; AT rich region)
- ρ factor-dependent
  - The amino-termina domain of ρ factor binds to hte RNA sequence of ρ utilization site
  - the carboxy-terminal domain of ρ hexameric ring closes
  - Ring closure propels ρ moving close to RNAP
  - ρ dis-engages RNA and RNAP from DNA

Eukaryotic Transcriotion

Three types of Nuclears RNAPs
- I: rRNA (28S, 18S)
- II: mRNA; snRNAs; miRNAs
- III: tRNAs; 5S rRNA; snRNA U6 7S RNA; other stable short RNAs
Transcription
- Subunit compositions of nueclear RNAPs
  - β’; β; αI; αII; ω
  - All yeast RNAP has five subunits similar to Bacteria
- RNAP II
  - Yeast RNAP II strcture resembling bacterial RNAPs
    - A vrab claw-like strcuture
- RNA biosynthesis
  - The clamp of the Rpb1 subunit moves down to trap DAN between the two claw
  - Unwind DNA at the active site
  - Wall of Rpb2 kinks the template by 90° out of the active site
  - One vase of the template points at the active site
  - This base is paired with the ribonucleotide
- RNA translocation
- Transcription promoters
  - Class I, II, II to RAN poly I, II, III
- Mammalian Gene and proximal promoter (Class II)
  - Proximal region (-200 ~ -30), within 200 bp of transcription start; similar to the -10; -35 in the bacteria’s
  1. TATA Box (TATAAA)
  - Recruits TATA binding protein …
  1. Initiator region (Inr)
  2. Other activation elements
- EPromoter and Activator proteins
  1. Transcriptional Activators: DNA binding domain; activation domain;
  2. Activator functions: Chromatin remodeling (acetylation-HAT); Mediator facilitates PIC assembly
- Repressor
  - Bind UAS.enhancers → displace activators
  - Prevent mediator → no PIC formation
  - Attracts HDACs and HMTs → heterochromatin
- PIC Assmbled
  - mediator facilitates TBP and TFIIB binding promoter
  - Other basal TFs and Pol II bind
  - phosphorylation of the CTD of Pol II by TFIIH → Transcription initiation
- Six transcription factors for clas II promoters (Not important)
- ==Phospho-code on RNAP II CTD
  - TFIH: Phosphate the Ser5 would initiated the transcription activity
  - P-TEFb: phospholate Ser2 to maintain the elongation state
- Non-canonical functions of transcription
  - CENP-A defines centromeric chromatin
- RNA…
- ChiP-Seq: Determine protein distributed on chromatins
- RNA-Seq: Expression profiles

Transcription inhibition as a therapeutic target for cancer

mRANs of many oncogenes
Transformed → RNA-directed agents: different sensitivity.

Alpha-amanitin: from mushroom
- directed binding to RNAP, adn block the bridge to repress the conformation change

Bacterial Transcription

Eukaryotic transcription

RNAP II CTD phospho-code

Non-canonical functions of transcription

Assays to study transcription

posttranscriptional Modification of Eukaryotic RNAs

Role of 5’ cap
- Ribosomal recogintion during translation
Cap structure
7-methylguanosin (m⁷G)
joined to the mRNA first nucleotide
Via a 5’-5’ tri-P brifge
Involved Enzymes
- RNA riphosphatase removing the γ-P of the mRNA’s 5’ site
- mRNA guanlyltransferase (a capping enzyme) adding GMP. (Take GMP to the site)
- Guanine-7-methyltranserase (second capping enzyme) methylating guanine
- Capping enzymes bind to RNAP-II, which will switch RAN synthesis initiation to elongation ()