AMB 11- RNA Structure, Processing, Translation, and Detection

Vocabulary practice flashcards covering eukaryotic mRNA processing, translation initiation, elongation, termination, non-coding RNAs, and quantitative protein measurement techniques.

Alternative Polyadenylation (APA)

A regulatory mechanism in which distinct polyadenylation sites are used in pre-mRNA processing, producing transcripts with alternative $3'\text{ UTR}$ sequences and diverse regulatory properties.

43S Preinitiation Complex

An early translation initiation complex composed of the $40\text{S}$ small ribosomal subunit bound to eukaryotic initiation factors (eIFs) and the initiator Met-$\text{tRNA}_i$.

Ternary Complex

A three-part translation initiation complex consisting of eIF2, GTP, and Met-$\text{tRNA}_i$ that delivers the initiator tRNA to the $40\text{S}$ ribosomal subunit.

eIF4F Complex

A heterotrimeric cap-binding initiation complex composed of the cap-binding protein eIF4E, the RNA helicase eIF4A, and the scaffolding subunit eIF4G.

eIF4E

The eukaryotic translation initiation factor responsible for specifically recognizing and binding the $5'$ $\text{m}^7\text{G}$ cap of mRNA.

eIF4A

An ATP-dependent RNA helicase subunit of the eIF4F complex that unwinds secondary structure in the $5'\text{ UTR}$ during small ribosomal subunit scanning.

eIF4G

A scaffolding subunit of the eIF4F complex that coordinates translation initiation by binding eIF4E, eIF4A, eIF3, and PABP, mediating crosstalk between the $5'$ cap and $3'$ poly(A) tail.

Poly(A)-Binding Protein (PABP)

A protein that binds the $3'$ poly(A) tail of eukaryotic mRNA and interacts with eIF4G to facilitate closed-loop communication between the $5'$ and $3'$ ends of the transcript.

eIF3

A multi-subunit eukaryotic initiation factor that bridges the cap-associated eIF4F complex to the $40\text{S}$ small ribosomal subunit.

eIF1 and eIF1A

Eukaryotic initiation factors associated with the $40\text{S}$ subunit that promote scanning and police accurate start codon (AUG) recognition and codon:anticodon base pairing.

eIF5

A GTPase-activating protein (GAP) that promotes GTP hydrolysis by eIF2 upon successful recognition of the AUG start codon.

eIF5B

A ribosome-dependent GTPase that bridges the $60\text{S}$ large ribosomal subunit to the $40\text{S}$ subunit to catalyze $80\text{S}$ initiation complex formation and initiation factor release.

Eukaryotic Elongation Factors (eEF1, eEF2)

Proteins that accelerate translation elongation and ensure fidelity by verifying codon:anticodon matching and catalyzing ribosomal translocation along the mRNA.

Eukaryotic Release Factors (eRF1, eRF3)

Proteins resembling tRNAs that recognize stop codons (UAA, UAG, UGA) within the ribosomal A site to terminate translation and release the completed polypeptide chain.

Immunoblotting (Western Blotting)

An analytical method used to measure specific protein levels by resolving denatured proteins by size using gel electrophoresis, transferring them to a membrane, and probing with target-specific antibodies.

Loading Control

A constitutively expressed housekeeping protein (such as GAPDH or Tubulin) monitored during Western blotting to verify equal sample loading across lanes.

Flow Cytometry

A laser-based biophysical technique used to quantify protein expression in individual cells within a liquid suspension as they pass single-file through a flow cell sensor.

ELISA (Enzyme-Linked Immunosorbent Assay)

A plate-based assay that utilizes target-specific antibodies and enzymatic or colorimetric detection to quantify specific protein concentrations in liquid samples.

Immunofluorescence (IF)

An optical detection technique utilizing fluorophore-conjugated antibodies to visualize the intracellular localization and spatial distribution of specific proteins.

Polyclonal Antibodies

A heterogeneous pool of antibodies obtained from immunized animal antiserum (e.g., rabbit) produced by multiple plasma B-cell clones that recognize multiple distinct epitopes on a target antigen.

Monoclonal Antibodies

A homogeneous population of identical antibodies produced by expanding single hybridoma cell clones that recognize a single, specific epitope on an antigen.

Bottom-Up Proteomics

A mass spectrometry-based strategy for global protein identification and quantification in which extracted proteins are enzymatically digested into peptides prior to ionization and mass-to-charge separation.

Small Nucleolar RNAs (snoRNAs)

Housekeeping non-coding RNAs localized in the nucleus that guide the chemical modification and processing maturation of pre-rRNAs.

Small Nuclear RNAs (snRNAs)

Housekeeping non-coding RNAs that assemble with proteins to form spliceosomal complexes involved in nuclear pre-mRNA splicing.

Circular RNAs (circRNAs)

Non-coding regulatory RNA molecules formed by backsplicing events that function in microRNA sponging, protein sponging, and signaling pathway modulation.

ILO 1- You can outline what a eukaryotic mRNA looks like, and how this differs from prokaryotic mRNAs

Eukaryotic mRNA is characterized by a 5' cap structure, a 3' poly-A tail, intron-exon organisation, and is monocistronic- encodes only a single protein/ polypeptide chain per molecule

Prokaryotic mRNA which typically lacks these features, has an SDS (Shine-Dalgarno sequence instead) and is usually polycistronic- (Carries code for multiple proteins on a single mRNA strand (often organized as operons); common in prokaryotes like bacteria, as well as mitochondria and chloroplasts)

ILO 2- You know how transcript variants can lead to regulatory complexity

Transcript variants arise from alternative splicing or alternative use of promoters and polyadenylation sites, resulting in different mRNA sequences that can produce diverse protein isoforms, thereby contributing to regulatory complexity in gene expression.

•Introns are spliced out in the nucleus, mostly co-transcriptionally

•Alternative splicing leads to protein diversity

Alternative poly adenylation (APA) site use can lead to alternative UTR sequences andregulatorydiversity

ILO 3- You know translation occurs in 3 phases (initiation, elongation, termination) 

Eukayotic mRNA translation:

The process involves three key phases: initiation, where the ribosome assembles on the mRNA; elongation, where amino acids are added to the growing polypeptide chain; and termination, where the completed protein is released upon encountering a stop codon.

•Most regulation is at the initiation phase

ILO 4- You know the steps of eukaryotic translation initiation, the key factors involved and what they do 

Eukaryotic translation initiation involves several key steps:

1- the assembly of the ribosome on the mRNA,

2- recognition of the 5' cap by eIF4E,

3- recruitment of the small ribosomal subunit with the help of eIF3, and scanning for the start codon, which is facilitated by eIF2 and AUG.

4- Key factors include eIF4F, eIF2, eIF3, and the 60S ribosomal subunit.

5- Ribosome finds 5’ cap and start codon via eukaryotic initiations factors (eIFs)

6- Movement from 5’ to 3’ end- Once the small ribosomal subunit (40S) docks at the 5' m7G cap, it moves directionally along the 5' UTR toward the 3' end until it encounters the correct AUG start codon.

ILO 5- You understand the methods we can use measure protein output

Measuring protein output globally-

To look at all proteins – use mass spectrometry

AKA “bottom-up proteomics”- Bottom-up proteomics is the standard mass spectrometry method used to identify and quantify proteins by digesting them into smaller peptides before analysis.

'“Top down proteomics” is when whole proteins are analysed - gain PTM insights, but usually have to enrich for or purify your protein of interest first

ILO 6- You know not all RNAs are messenger RNAs, and can provide examples

RNAs can have other important jobs/ structures:

•Transfer RNAs (tRNAs) and ribosomal RNAs (rRNA) function in translation

•Small nucleolar RNAs (snoRNAs) help with rRNA processing

•Small nuclear RNAs (snRNAs) help with splicing

•Small regulatory RNAs (microRNAs, siRNAs, piRNAs)

•Long non-coding RNAs (lncRNAs) – regulatory roles

•Circular RNAs (circRNAs) – formed from backsplicing

•Ribozymes – self-cleaving RNAs with regulatory roles

Detecting different protein isoforms:

1) Clever RT-PCR primer design, then gel electrophoresis. RT-PCR is a technique that uses an enzyme to turn single-stranded RNA into complementary DNA (cDNA), which is then amplified using standard PCR.

2)Transcript level short read RNA-seq analysis- measures the expression of individual mRNA isoforms (transcript variants) rather than just looking at the overall gene level, using short sequencing reads (typically 50–150 base pairs).

150-300bp reads

cDNA synthesis

PCR amplification

fragmentation

3) Long read RNAseq (eg PacBio/ Oxford Nanopore)-sequences entire, full-length transcripts in a single read (ranging from 1,000 to over 10,000 base pairs), completely bypassing the need to fragment the RNA.

•15-25kbp reads

• cDNA synthesis

4) Direct RNA Sequencing (Nanopore)-

sequences native, full-length RNA molecules directly as they pass through a nanopore, completely skipping the reverse transcription (RT) and PCR amplification steps.

Ribosome composition:

mass 2/3 RNA, 1/3 protein

large and small subunits

Prokaryotes have 70S ribosomes and eukaryotes have 80S ribosomes, which is based on the different sedimentation rates of these macromolecular complexes during high-speed centrifugation.

The Svedberg Unit (S): The "S" stands for a Svedberg unit, which measures the sedimentation rate of a particle when subjected to the high centrifugal force of an ultracentrifuge.

Ribosome finds 5’ cap and start codon via eukaryotic initiations factors (eIFs)

Step 1 of eukaryotic translation initiation1. Formation of the 43S Preinitiation Complex (40S+eIFs+Met-tRNAi)

eIF1 + 1a- proofreading AUG selection

eIF3- bridges eIF4F and ribosome

40S- small ribosomal subunit

eIF5- GTPase-activating protein for eIF2 (

eIF2- tRNAi-GTP

eIF2:GTPase

Ternary complex (i.e. made of 3 things)

tRNA delivery

2. Recruitment of the small ribosomal subunit (43 Preinitiation Complex) to the mRNA cap

eIF4F complex on the mRNA cap:

• eIF4G-scaffolding subunit

• eIF4A- helicase

• eIF4E- cap binding protein

• PABP- Poly A binding protein (Crosstalk between 5’ and 3’ end)

3. Small ribosomal subunit scanning to find the start codon

5’-3’ scanning to find AUG requires:

Unfolding of 5’UTR

ATP hydrolysis (helicase) VIA EIF4A

4. Start codon recognition

GTP hydrolysis

tRNA delivery

eIF1 polices accurate codon:anti-codon matching

When the small ribosomal subunit successfully recognises and base-pairs with the AUG start codon, eIF5 triggers GTP hydrolysis on eIF2 (converting eIF2-GTP to eIF2-GDP).

5.80S formation (ie 60S joining)

eIF release

large 60S subunit joining

60S- large ribosomal subunit

peptidyl transfer centre

eIF5B- GTPase

Bridges the 60S to the 40S

GTP hydrolysis and (2^nd) eIF release

Elongation:

elongation factors (eEF1, eEF2) accelerate translation elongation and ensure accuracy of codon:anticodon matching

Each amino acid added to the growing end of a polypeptide chain is selected by complementary base-pairing between the anticodon on its attached tRNA molecule and the next codon on the mRNA chain.

Because only one of the many types of tRNA molecules in a cell can base-pair with each codon, the codon determines the specific amino acid to be added to the growing polypeptide chain.

The four-step cycle shown is repeated over and over during the synthesis of a protein.

Step 1, an aminoacyl-tRNA molecule binds to a vacant A site on the ribosome.

Step 2, a new peptide bond is formed. In

step 3, the large subunit translocates relative to the small subunit, leaving the two tRNAs in hybrid sites: P on the large subunit and A on the small, for one tRNA; E on the large subunit and P on the small, for the other tRNA.

Step 4, the small subunit translocates carrying its mRNA a distance of three nucleotides through the ribosome.

This “resets” the ribosome with a fully empty A site, ready for the next aminoacyl-tRNA molecule to bind. eEfs expend energy (GTP) if they have to remove a mismatched tRNA. elongation very similar in euks and prots.

Termination:

release factors (eRF1, eRF3) resemble tRNAs, recognise stop codons and terminate translation

Peptide release, separation of small and large subunits. Very similar in euks and prots

Immunoblotting (aka western blotting) with protein- specific antibodies

This is a technique used to detect specific proteins in a sample by separating proteins via gel electrophoresis, transferring them to a membrane, and probing with antibodies that specifically bind to the target protein.

Loading control- ‘housekeeping gene’ on the gel

Flow Cytometry (“flow”)- Fluorescence-activated Cell Sorting (FACS)-

(Suspends single living cells in a fluid stream and passes them through lasers to rapidly count and analyze their physical traits, internal complexity, and surface markers based on light scattering and fluorescence).

ELISA (enzyme-linked immunosorbent assay) also work using specific antibodies

Uses specific antibodies fixed to a multi-well plate to capture and quantify the exact concentration of proteins, hormones, or antibodies in liquid samples e.g. serum through a color-changing chemical reaction.

Immunofluoresence(IF) uses specific antibodies to detect intracellular localisation of proteins

(Uses fluorescently labeled antibodies to bind specific target proteins directly within intact fixed tissues or cells, allowing scientists to visualize their exact spatial location and structural distribution under a microscope.)

Commercial antibodies for research-

Can also take the antibody producing cells from inoculated animal, typically mice, fuse with cancer cells so grow exponentially, and grow in culture.

Identify the one making the best antibody, and culture that so it secretes antibody, no more animals involved. Can also sequence the gene that makes the antibody heavy+long chain and then make the antibody synthetically.

The future – designer antibodies for Tx and research-

• Modular, AI-engineered antibodies customized to hit highly specific disease and research targets.

• Therapeutics (Tx): Deliver targeted chemotherapy (ADCs), bridge T-cells to tumors (Bispecifics), and cross the blood-brain barrier.

• Research: Track/block proteins inside living cells (Intrabodies) and enable ultra-high-resolution imaging.

• Production: Designed entirely from scratch in silico using AI instead of animals.