Unit 4

4 Ribosomes

All three types of RNA are involved in translation/ making proteins

Ribosomes

  • Highly abundant compact particles (number will vary depending on  cell function but a human cell can have up to 10 million ribosomes)

  • Discovered by George Palade in 1953 who observed them through electron microscopy (known as particles of palade)

  • Present in teh cytosol of all cells except mature spermatozoon and in RBCs where they are very scarce

  • Essential for protein synthesis

  • Composed of two distinct subunits (large and small) but they have different size in eukaryotes (80S) and prokaryotes (70S)

  • Svedbger units (S): sedimentation rate of particles that have been subjected to centrifugation. Depends on size, shape and density

don't need to know amount of proteins

Ribosomes are found in:

  • Cytosol (liquid inside plasma membrane outside nucleus) (cytoplasm liquid and organelles)

  • Attached to RER

  • Attached to outer nuclear membrane

  • Inside the mitochondria or the chloroplasts

Ribosomal biogensis

Ribosome subunits are assembled in the nucleolus

Ribosomal proteins and rRNAs are synthesized in different compartments.

  • Ribosomal Proteins are synthesized in the cytosol and then enter the nucleus through nuclear pores

  • Nucleolar DNA is transcribed to produce rRNA that will be subsequently processed

  • Ribosomal proteins and RNAs assemble in the nucleolus and form the small and large ribosome subunits

  • The two subunits exit the nucleus through nuclear pores. Functional ribosomes will be

formed in the cytosol

Transfer RNA (tRNA)

The tRNA molecule has a characteristic cloverleaf structure with three hairpin loops

  1. One of these hairpin loops contains a three nucleotide sequence called the ANTICODON, that can recognize and decode a CODON sequence in mRNA.

  2. At the 3 ́end, each tRNA has its corresponding amino acid attached through an ester bond. There is at least one specific tRNA for each one of the 20 amino acids found in proteins

tRNAs read the information encoded in mRNA and transfer the appropriate amino acid to the nascent polypeptide chain during protein synthesis.

The mRNA sequence is decoded in sets of three nucleotides or codons according to the genetic code

Code is redundant: most amino acids are specified by more than one triplet

There is a unique AUG codon that acts as initiation codon for the synthesis of all proteins 

There are three codons that do not code for any amino acid they are stop codons

tRNA and amino acid activation

Tyhe correct pariing of each tRNA to its amino acid occurs in the cytoplasm and is catalysed by aminoacyl-tRNA synthetases

Amino acid + tRNA = ATP → aminoacyl-tRNA + AMP + PPi

  • A high energy ester bond is formed between the 3 ́OH of the tRNA and the appropriate amino acid 

  • This reaction requires ATP and involves the formation of an activated amino acid-AMP intermediate

  • There are many different aminoacyl-tRNA synthetases (one for each amino acid and corresponding tRNA)

Translation

Translation is always initiated on cytosolic ribosomes regardless of the nature of the translated protein.

The process of translation occurs in three stages:

  1. Initiation

  2. Elongation

  3. Termination

Activation of amino acids (formation of aminoacyl-tRNA) must occur before translation, and protein maturation and folding will take place after translation

Translation initiation

Translation initiation involves the formation of a RIBOSOME TRANSLATION INITIATION

COMPLEX where the anticodon in the initiator tRNA (Met-tRNAiMet) pairs with the start

codon in the mRNA molecule.

  1. The Small Ribosome Subunit binds to the mRNA molecule and finds the Start Codon

  2. The first tRNA, the Initiator tRNA (tRNAi-Met ) enters the P site and pairs with the start AUG codon

  3. The Large Ribosomal Subunit assembles with the small subunit to form the ribosome Translation Initiation Complex

Initiator tRNA
- the initiator tRNA is different from other tRNAs (it has a different sequence)

The initiator tRNA is not the same in eukaryotes (methionine-tRNAi) and in prokaryotes (Formyl-Methionince-tRNAi)

All newly synthesized polypeptides start with the amino acid methionine in eukaryotes and formyl-methionine in prokaryotes

Identification of the start codon

  • In prokaryotes: Start codons in bacterial mRNAs are preceded by a specific sequence known as the SHINE-DALGARNO SEQUENCE . This sequence is located a few nucleotides upstream of the initial AUG and is recognized by the 3’end of the 16S ribosomal RNA. This binding helps align the mRNA on the ribosome for translation initiation.

  • In eukaryotes: Ribosomes bind to the 5 ́CAP (Methyl Guanosine Triphosphate) of mRNAs and scan the mRNA sequence downstream of the cap until they find an AUG that fits with the KOZAK consensus sequence : ACCAUGG . The sequence between the 5’ end and the start codon is not translated and represents the 5’ untranslated region (5’ UTR) of the mRNA

  • The basic mechanism is similar to that of prokaryotes but more complex, with at least 10 Initiation Factors (eIFs) involved

  • One of the initiation factors helps recognize the 5’ cap in mRNA

  • The final Initiation Complex is similar except with Met-tRNAi and with a larger ribosome (80S)

  • In eukaryotes the 5’ Cap (Met-G- 3P) interacts with the 3’ poly A tail through the poly-A binding protein (PABP) and some initiation protein complexes. They form a loop that is maintained throughout the translation process so that when the ribosome reaches the termination site it is close to the 5’ end and can initiate another round of translation

Translation elongation

Similar in prokaryotes and eukaryotes and involves several elongation factors

  1. Binding of aminoacyl-tRNA to locus A

  2. Formation of peptide bonds 

  3. Translocation of the ribosome

  1.  Binding of aminoacyl-tRNA to locus A

  • The initiator tRNAi is bound at the P site. The first step in elongation is the binding of the next aminoacyl-tRNA to the A site by pairing with the second codon

  • The aminoacyl-tRNA is escorted to the A site by the Tu elongation factor (EF-Tu) that is complexed with GTP 

  • Correct pairing of the codon in mRNA and the anticodon in the tRNA induces hydrolysis of the EF-Tu-bound GTP and release of the elongation factor

  1. Formation of peptide bonds 

  • A peptide bond is formed between the amino acid bound to the initiator tRNA (f-Met or Met) and the amino acid bound to the tRNA on the A site. The Methionine is transferred to the aminoacyl-tRNA on the A site

  • The newly formed dipeptide stays bound to the tRNA on the A site while the P site is now occupied by an uncharged, amino acid-free, initiator tRNAi

  • The energy required for peptide bond formation is provided by the breakage of high energy bond between the amino acid and the tRNA (established during the amino acid activation)

  • The condensation reaction is catalysed by an rRNA with enzymatic activity (peptidyl-transferase) in the large ribosomal subunit: 23S or 28S rRNA

  1. Translocation of the ribosome

  • Translocation of the ribosome involves another GTP- bound Elongation Factor and requires GTP hydrolysis.

  • GTP hydrolysis allows the ribosome to slide down to the following codon (towards the 3’ end ) and change the position of the peptidyl-tRNA (tRNA-aa-Met) from the A site to the P site.

  • The uncharged, amino acid-free, initiator tRNAi is now sitting on the E site and the A site is empty.

  • The binding of a new codon-matching tRNA to the A

site will induce the release of the tRNAi on the E site

P = Peptidol

A = Aminoacyl

E = Exit site

Translation termination

  •  The three STOP CODONS (UAG, UAA or UGA) are non- sense codons that have no matching tRNAs with complementary anticodons

  • When a stop codon reaches the A site on the ribosome, it is recognized by a Release Factor rather than a tRNA

  • The Release Factor (RF or eRF-1) stimulates hydrolysis of the bond between the tRNA on the P site and the polypeptide chain 

  • The completed polypeptide is released from the ribosome

  • The tRNA is discharged and the mRNA and the two ribosomal subunits dissociate

  • In eukaryotes the loop structure formed by the mRNA (association of the 5’ and 3’ends ) facilitates the reassociation of the ribosome subunits at the 5’ end and the initiation of a new translation cycle

Polysomes or polyribosomes

Each mRNA molecule can be simultaneously translated by multiple ribosomes forming a polysome or polyribosome (in prokaryotes and eukaryotes)

Once the first ribosome moves away from the initiation site, another ribosome can bind to the same mRNA and start the synthesis of a second polypeptide chain

Each ribosome in the polysome functions independently from the others and synthesizes a separate polypeptide chain

This strategy significantly speeds up the overall rate of protein synthesis and makes the process much more efficient

Ribosomes and protein synthesis as therapeutic targets

  • Many of our most effective ANTIBIOTICS are compounds that act by inhibiting bacterial, but not eukaryotic, protein synthesis. This specificity is critical to avoid toxicity on human cells

  • Inhibiting bacterial protein synthesis will inhibit bacterial growth and proliferation

  • STREPTOMYCIN, CHLORAMPHENICOL, TETRACYCLINE and ERYTHROMYCIN, are specific for prokaryotic ribosomes and are therefore very effective as antibacterial drugs.

  • Other antibiotics such as PUROMYCIN OR CYCLOHEXIMIDE are not used as therapeutic agents because they act on both prokaryotes and eukaryotes or specifically on eukaryotes

Proteasomes

A Proteasome is a eukaryotic multisubunit protease complex that degrades proteins that have been marked for destruction by a small protein called UBIQUITIN. After the first Ubiquitin molecule has been covalently attached to the protein that will be degraded, additional Ubiquitins are linked to the first one forming a poly-ubiquitin chain that is recognized by the proteasome

Proteasomes are found in the cytosol and the nucleus

The timed and controlled degradation of proteins is an essential mechanism to regulate the abundance of specific proteins in the cell

The Ubiquitin-Proteasome pathway plays an important role in several biological processes:

  • Quality control of proteins

  • Cell cycle regulation

  • Elimination of misfolded proteins (particularly relevant in neurodegenerative disorders such as Huntingtons or Alzheimers)

Structure of the proteasome

The Proteasome complex shows a barrel- like structure with a catalytic nucleus (20S) made of 4 piled rings with protease active sites in the central pore

Two additional outer rings act as entry gates for the proteins to be degraded or as exit gates for the resulting small peptides or amino acids