Stages and Regulation of Translation

Translation is the process of synthesizing a protein from a messenger RNA (mRNA) transcript. This process is divided into three primary stages: initiation, elongation, and termination. Translation is catalyzed by structures known as ribosomes, which are large complexes of proteins and ribosomal RNA (rRNA). The ribosome “reads” the mRNA, and brings in transfer RNAs (tRNAs), each bound to a specific amino acid, in the correct order. These amino acids are then bound to one another by an enzymatic component of the ribosome known as peptidyl transferase. Translation can be regulated at multiple steps, including through a process known as RNA interference. This process involves small segments of double-stranded RNA which are able to inhibit translation of mRNAs.

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Central Dogma and Genetic Code

Definition

  • The central dogma of gene expression: To express a gene, DNA is transcribed into RNA, which is then translated into a polypeptide.
  • Translation is the process by which messenger RNA (mRNA) is used as a template to make polypeptides.

DNA

  • A double-helix molecule made of 2 antiparallel strands, with a structure similar to a twisted ladder:
    • The “sides” of each ladder are made up of alternating deoxyribose (a 5-carbon sugar) and phosphate molecules.
    • The “rungs” of the ladder are made of matched nitrogen-containing molecules called nucleotides, frequently referred to as “bases.”
  • DNA base pairs:
    • Guanine (G), cytosine (C), adenine (A), and thymine (T)
    • G pairs with C (and vice versa) via 3 hydrogen bonds.
    • A pairs with T (and vice versa) via 2 hydrogen bonds.
    • These base pairs can be “read” as a string of letters; e.g., GTATCGA.
    • This string of letters is the “code,” or instruction manual, that is ultimately used to create proteins.

RNA

  • A single-stranded molecule made up of alternating ribose (a 5-carbon sugar) and phosphate molecules
  • Each ribose is bound to an RNA nucleotide:
    • Guanine (G), cytosine (C), adenine (A), and uracil (U)
    • Note that instead of thymine, adenine binds with uracil (and vice versa) via 2 hydrogen bonds.
  • Codon: a series of 3 nucleotides in a row that code for a particular amino acid
  • Types of RNA:
    • mRNA: template strands that are translated into polypeptides by the ribosomal complexes
    • Ribosomal RNA (rRNA): a component of the ribosome complex that assists in protein synthesis.
    • Transfer RNA (tRNA): molecules that carry amino acids to the ribosome, where they bind to the mRNA
Transcription Nucleic-acids

Structures of RNA and DNA

Image by Lecturio.

The genetic code

The genetic code is how organisms translate a sequence of bases into an actual protein.

  • Information in the mRNA is contained in 3-base sequences called codons.
  • The code is specific: each codon codes for only 1 amino acid.
  • The code is redundant: 
    • With 4 bases, there are 64 possible 3-base codons, but only 20 amino acids.
    • Some amino acids may be coded for by several different codons:
      • Usually it is the 3rd base that differs (known as the wobble base).
      • Mutations in the wobble base are often silent mutations that do not affect the amino acid sequence.
      • For example, GGG, GGA, GGC, and GGU all code for glycine, so even if GGG mutates to GGA, the final protein will be the same.
  • The code contains “punctuation”:
    • Start codon (initiates translation): AUG → codes for methionine
    • Stop codons (terminate translation): UAA, UAG, UGA
  • The code is universal: 
    • The code is the same in all species, including prokaryotes and eukaryotes.
    • Exception: several codons are slightly different within mitochondria.
  • The code is read by tRNAs inside the ribosomes.
Genetic Code

The genetic code:
Start in the center and read out to determine for which amino acids each of the 64 codons codes. Start and stop codons are noted.

Image by Lecturio.

Components of Translation

mRNAs are translated into proteins by the ribosomes and transfer RNAs.

Transfer RNAs (tRNAs)

tRNAs carry amino acids to the ribosomes, where they bind to the mRNA, lining up the amino acids that will bond to form the growing polypeptide.

  • Synthesized by:
    • RNA polymerase in prokaryotes
    • RNA polymerase III in eukaryotes
    • There is a unique tRNA for each amino acid.
  • Structure: 
    • A single-stranded RNA molecule is 70–85 nucleotides in length.
    • Forms hydrogen bonds/base pairs with itself to create cloverleaf (secondary) structure
    • 3’ end (on the stem of the clover) is a binding site for an amino acid.
    • Middle loop of the clover contains an anticodon: 
      • A sequence of 3 bases that are complementary to an mRNA codon.
      • Allow the tRNA to bind to an mRNA where the codon and anticodon match
  • tRNA charging: 
    • The process of connecting an amino acid to the tRNA
    • Catalyzed by the enzyme aminoacyl-tRNA synthetase
  • Aminoacyl-tRNA synthetase: 
    • Responsible for connecting the correct amino acid to the correct tRNA
    • There is a different aminoacyl-tRNA synthetase for each amino acid/tRNA.
    • Contains 2 primary binding sites:
      • tRNA binding site: includes a codon-like structure that binds to the tRNA’s anticodon
      • Amino acid binding site: fits the amino acid that matches that anticodon
    • Charging process:
      • Amino acid loaded in its binding site 
      • ATP is cleaved, creating an AMP to charge the amino acid.
      • tRNA is loaded into its binding site.
      • Amino acid is paired to tRNA, while the AMP is released.
      • Charged tRNA is released from the enzyme.
    • The amino acid bound at the 3’ end of the tRNA will match the codon on the mRNA to which the tRNA anticodon can bind.
  • Initiator tRNAs: the tRNAs that pair with the start codon
    • In prokaryotes, the initiator tRNA is N-formylmethionine (fMet)-tRNA
    • In eukaryotes, the initiator tRNA is methionine-tRNA (met-tRNAi)

Ribosomes

The ribosomes are catalytic complexes that include both protein and rRNA components. Within the ribosomal complex, the mRNA is read by tRNAs and a polypeptide is created.

  • Ribosome structure:
    • Made up of 2 primary subunits: a large subunit and a smaller subunit
    • Contains both multiple proteins and rRNAs
    • rRNAs form extensive secondary structures by pairing with themselves.
  • Large subunit:
    • Contains peptidyl transferase:
      • A ribozyme (an rRNA that functions as an enzyme to catalyze a reaction): the largest rRNA within the ribosome.
      • Creates the peptide bonds between amino acids
    • Contains 3 binding sites for charged tRNAs:
      • Arrival (A) site
      • Polypeptide (P) site
      • Exit (E) site
  • Small subunit: decodes the mRNA
  • Ribosomes read mRNA from 5’ to 3’ 
  • Located floating free within the cytosol or can attach to the rough endoplasmic reticulum (RER)
Ribosome structure

Ribosome structure demonstrating the large subunit on top, with the A, P, and E binding sites for charged tRNAs. The smaller subunit is below the mRNA.

Image by Lecturio.
Table: Prokaryotic versus eukaryotic ribosomes
ProkaryotesEukaryotes
Size of the small subunit30 S40 S
Size of the large subunit50 S60 S
Number of proteins5288
Number of rRNAs34
Size of homologous rRNAs in the small subunit16 S18 S
Sizes of homologous rRNAs in the large subunit
  • 5 S
  • 23 S*
  • 5 S
  • 28 S*
Size of rRNA in the large subunit without a prokaryotic homologue5.8 S
* The rRNA known as peptidyl transferase catalyzes the formation of peptide bonds between amino acids in the growing polypeptide chain.

Initiation of Translation

Initiating translation involves assembling the ribosome on the mRNA in the proper direction and finding the start codon.

Ribosome assembly

  • Ribosomal subunits are disassembled in the cytosol when not in use.
  • The small subunit of the ribosome attaches to the 5’ end of the mRNA.
  • The large subunit does not attach until after the initiator tRNA binds to the start codon.
  • In prokaryotes:
    • The ribosome assembles on the mRNA as it is being transcribed from DNA.
    • No cap is present or required to determine directionality.
  • In eukaryotes:
    • The mRNA must be transported from the nucleus, where it was transcribed, to the cytosol for translation.
    • mRNAs have a 5’ cap, which:
      • Indicate directionality of the mRNA
      • A binding site for eukaryotic initiation factors (eIFs)
    • Multiple eIFs are required to help the small subunit and initiator tRNA bind the mRNA.
  • Binding of the eIFs and ribosomal subunits requires energy.

Scanning for the start site

Once the small subunit has bound to the 5’ end of the mRNA, the small subunit begins scanning for the start site.

  • The start site: AUG (the codon for methionine)
    • Prokaryotes: use a specific purine-rich sequence on the 5’ end to distinguish the start AUG from other (internal) AUGs
    • Eukaryotes: use the first AUG encountered nearest the 5’ end
  • Ribosomal complex scans the mRNA for the start codon (AUG) by moving step by step in the 5’ to 3’ direction along the mRNA. This process requires ATP hydrolysis for energy to move.
  • When the small subunit reaches the start codon (AUG) on the mRNA, the mRNA will bind to an initiator tRNA.
  • Once the initiator tRNA is bound to the mRNA, the large subunit will come in and orient itself so that the initiator tRNA is located in the P-site of the complex.
  • Additional eIFs and energy are required in eukaryotes to fully assemble the ribosome.
Assembly of a ribosome

Assembly of a ribosome
fMet: formylmethionine

Image by Lecturio.

Elongation and Termination of Translation

Process of elongation

  • An amino acid–charged-tRNA (aa-tRNA) binds to the A-site within the ribosome:
    • The tRNA anticodon must complement the mRNA codon in order for binding at the A-site to occur.
    • Requires energy from an elongation factor (EF) hydrolyzing a GTP 
    • The specific EFs are:
      • eEF-1α in eukaryotes
      • EF-Tu in prokaryotes 
  • Peptidyl transferase connects the new amino acid to the growing polypeptide chain by:
    • Transferring the polypeptide chain from the tRNA in the P-site to the amino end (“top”) of the amino acid connected to the tRNA in the A-site
    • Catalyzing formation of a peptide bond between the 2 amino acids
  • The ribosome translocates 1 codon further in the 3’ direction:
    • Translocation requires energy from another EF hydrolyzing a GTP:
      • EF-G in prokaryotes
      • eEF-2 in eukaryotes
    • This translocation shifts the tRNAs:
      • The tRNA in the A-position with the growing polypeptide chain moves to the P-position.
      • The tRNA in the P-position moves to the E-site and is ejected from the ribosome.
      • The A-site is now open for the next charged tRNA to enter.
  • This cycle is repeated until a stop codon is encountered.

Wobble pairing

  • Rules for base pairing are not as stringent in the 3rd position.
  • Wobble pairing: when the base in the 3rd position of the codon does not match the base in the 3rd position of the anticodon
  • Many amino acids have multiple possible 3rd bases, where wobble pairing would not alter the final structure of the protein.
  • If wobble pairing allows the insertion of a different amino acid, a mutant protein may result.
Wobble pairing

Wobble pairing

Image by Lecturio.

Formation of peptide bonds

  • Catalyzed by peptidyl transferase (an enzymatic rRNA within a ribosome)
  • Bonds the α-carboxyl carbon to the α-amine nitrogen
  • Releases H2O in the process
Formation of a peptide bond between 2 amino acids

Formation of a peptide bond between 2 amino acids

Image by Lecturio.

Termination of translation

Termination occurs when the ribosome reaches a stop codon.

  • Stop codons code for a release factor (RF) rather than amino acids.
  • The RFs enter the A-site and cause:
    • The ribosome to disassemble
    • Release of the polypeptide chain
Termination of translation

Termination of translation

Image by Lecturio.

Regulation of Translation

Translation can be regulated at the level of initiation, elongation, or termination, primarily through up-regulation and down-regulation of initiation, elongation, and termination factors. Translation is further regulated through RNA interference, alternative splicing, and RNA editing. 

RNA interference

RNA interference (RNAi) is interference in translation by small double-stranded RNA molecules that ends up inhibiting translation of specific mRNAs.

  • Involves double-stranded RNAs and a protein complex:
    • MicroRNAs (miRNAs): originate within the cell
    • Silencing (or small interfering) RNAs (siRNAs): originate outside the cell 
      • May originate from a virus 
      • Commonly used in biotechnology
    • RNA-induced silencing complex (RISC): complex of proteins that incorporates a miRNA or a siRNA and can inhibit translation
  • siRNAs and miRNAs are involved in:
    • Selective degradation of mRNA
    • Inhibition of translation 
    • Alteration of chromatin structure (epigenetic mechanisms)
  • How RNAi works:
    • An enzyme called dicer cleaves double-stranded RNA (dsRNA) into approximately 20 base-pair segments (either miRNA or siRNA).
    • Once diced, the dsRNA is separated into single strands.
    • When these single-stranded miRNA/siRNA segments bind to RISCs, the RISC assists the miRNA/siRNA in binding to and inhibiting a complementary mRNA strand.
    • Once bound to an mRNA, the RISC inhibits translation by:
      • Cleaving mRNA segments with the enzyme argonaute (part of the RISC).
      • Remaining bound to the mRNA and blocking the ribosome from completing translation.
  • Purposes of RNAi:
    • Protection against certain viruses
    • Regulation of gene expression
    • In biotechnology: allows molecular biologists to silence specific target genes
RNA interference via the RISC and a miRNA

RNA interference via the RISC and a miRNA

Image by Lecturio.

Alternative splicing

  • Non-coding introns are spliced out by spliceosomes (enzymatic ribonucleoprotein complexes).
  • Multiple different proteins can be made from a single gene with differential splicing of the mRNA.
Alternative splicing

Alternative splicing:
By splicing it in different ways, different proteins can be created from the same mRNA.

Image by Lecturio.

RNA editing

  • Enzymes can edit the mRNA transcripts after they’ve already been created.
  • Examples of proteins whose mRNAs undergo RNA editing: 
    • Apolipoprotein B
    • Serotonin receptors

Clinical Relevance

  • Macrolides and ketolides: a group of antibiotics commonly used in respiratory infections which work by inhibiting the 50S subunit on the ribosome, blocking protein synthesis in the bacteria. Common macrolides include erythromycin, clarithromycin, and azithromycin.
  • Tetracyclines: a group of broad-spectrum bacteriostatic antibiotics which work by inhibiting the 30S subunit on the ribosome, blocking protein synthesis in the bacteria. A common example is doxycycline.
  • Diphtheria toxin: diphtheria toxin ribosylates eEF-2 thereby inhibiting elongation, and thus protein synthesis, leading to cell death. The characteristic findings of diphtheria include pharyngeal pseudomembranes (grayish tonsillar exudates), severe pharyngitis and a “bull’s neck” lymphadenopathy. Treatment is primarily through passive immunization with antitoxin and antibiotics. Prevention is via the diphtheriae toxoid vaccine.
  • Cancer: miRNAs can act as either tumor suppressors or oncogenes by dysregulating gene expression. 

References

  1. Weil, P. A. (2018). Nucleic acid structure & function. In Rodwell, V.W., et al. (Eds.). Harper’s illustrated biochemistry, 31e. New York, NY: McGraw-Hill Education. Retrieved from https://accessmedicine.mhmedical.com/content.aspx?aid=1160190679
  2. Weil, P. A. (2018). Protein synthesis & the genetic code. In Rodwell, V.W., et al. (Eds.). Harper’s illustrated biochemistry, 31e. New York, NY: McGraw-Hill Education. Retrieved from https://accessmedicine.mhmedical.com/content.aspx?aid=1160191039
  3. Cooper, G. Hausman, R. (2013). The Cell: a molecular approach. Sunderland, MA: Sinauer Associates.
  4. Berg, J.M., Tymoczko, J.L., Stryer, L. (2002). Section 29.5, Eukaryotic Protein Synthesis Differs from Prokaryotic Protein Synthesis Primarily in Translation Initiation. In Biochemistry, 5th edition. New York: W H Freeman. Retrieved April 19, 2021, from  https://www.ncbi.nlm.nih.gov/books/NBK22531/

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