Class 12 Slides

The “reading frame” is set during

translation initiation and maintained during elongation

Why can’t the ribosome fix incorrectly-added amino acids?

• That’s not how translation works

• Ribosomes facilitate tRNA-mRNA base pairing and peptide bond formation

• aaRS enzymes match tRNAs to aminio acids

Open Reading Frames can be formed by

splicing or can exist as exons independently

ways translation is regulated

1. translation prevented before it begins by lack of eIF2-GTP

2. translation block via RISC binding

3. translation of an upstream ORF instead of the protein-coding ORF

4. alternative start codons create variants of the protein

eIF2 is Important for

Translation Initiation

eIF2 step 1

small ribosomal subunit with initiator tRNA bound to P site

eIF2 step 2

additional initiation factors

eIF2 step 3

initiator tRNA moves along RNA searching for first AUG

eIF2 is Important for 4

eIF2 and other initiation factors dissociate

Translation is regulated

Regulation via upstream start codons

can be global or transcript specific.

Global

low [aa-tRNAs] favors skipping the first start codon

Specific

mRNA structure/folding can influence where the ribosome assembles,

proteins may bind to specific mRNAs to block initiation at one or more start codons

Ways translation is regulated

translation of an upstream ORF instead of the protein-coding ORF

alternative start codons create variants of the protein

How/Why do uORFs stop or prevent translation? 1

Ribosomes that are translating uORFs are not available to translate other ORFs

How/Why do uORFs stop or prevent translation? 2

Peptides can start to fold in the ribosome exit tunnel, disrupting the structure of the ribosome and causing it to stall

How/Why do uORFs stop or prevent translation? 3

Ribosomes at “premature” stop codons cause ribosomes to stall

Release Factors

work most efficiently when close to the poly-A tail

Stalled ribosomes

Stalled ribosomes block upstream ribosomes from completing translation of the peptide

Stalled ribosomes cause degradation of the mRNA via nonsense- mediated decay

Nonsense-mediated decay of mRNA

Nonsense codons = stop codons

• Stop codons that occur that are not close to the 3’ end OR are upstream of splice junctions

Nonsense-mediated decay of mRNA

premature stop codon, transcription errors or mutation during DNA replication can cause early stop codons in the mRNA

Exon junction complexes stay associated with splice sites after mRNA processing

normal translation

Ribosome binds near 5’ end

Translation starts at the start codon

Release Factors bind when ribosome reaches the stop codon

Translation terminates, releasing the protein, RFs, and ribosomal subunits

translation of an mRNA with a premature stop codon:

Ribosome binds near 5’ end

Translation starts at the start codon

Release Factors bind when ribosome reaches the premature stop codon

When RFs bind upstream of an EJC or far from the 3’ end, UPF proteins bind

Translation terminates, releasing the protein, RFs, and ribosomal subunits

UPFs recruit endonucleases, uncapping proteins, deadenylation proteins

The mRNA is uncapped, deadenylated, cut, degraded

translation of an mRNA with a premature stop codon 1

Ribosome binds near 5’ end

translation of an mRNA with a premature stop codon 2

Translation starts at the start codon

translation of an mRNA with a premature stop codon 3

Release Factors bind when ribosome reaches the premature stop codon

translation of an mRNA with a premature stop codon 4

When RFs bind upstream of an EJC or far from the 3’ end, UPF proteins bind

translation of an mRNA with a premature stop codon 5

Translation terminates, releasing the protein, RFs, and ribosomal subunits

translation of an mRNA with a premature stop codon 6

UPFs recruit endonucleases, uncapping proteins, deadenylation proteins

translation of an mRNA with a premature stop codon 7

The mRNA is uncapped, deadenylated, cut, degraded

uORFs are common in

animal genomes

• About half of human genes include potential uORFs

• Some mRNAs have multiple uORFs and a “real” protein-coding ORF

This is another layer of regulation that can

prevent inappropriate or excessive gene expression

• chromatin • mRNA processing

• DNA methylation • export from the nucleus

• DNA looping • selection of the protein-coding AUG

• sequence-specific TF binding • mRNA stability

Ribosome profiling revealed the existence of, Key Points from a 2017 review by Cuoso and Patraquim

smORFs

• Small peptides of 100 amino acids or fewer are encoded by small open reading frames (smORFs) and mediate key physiological functions in animals and humans.

• smORFs constitute 99% of transcribed, but only 1% of annotated, coding sequences in flies, mice and humans.

• Different smORF classes show distinctive and predictive markers of functionality at the RNA level and the protein sequence level.

• The characteristics of different smORF classes are evolutionarily conserved across animal species, encouraging the use of Drosophila melanogaster and Mus musculus as model organisms for studies of peptide biology in the context of development, physiology and disease.

• Different smORF classes may represent steps in the origin and evolution of new genes and proteins.

Ribosome profiling revealed the existence of

smORFs

Peptides encoded by smORFs are functional

• bind to and regulate other proteins

• localize specifically to organelles and function there