Protein Synthesis, Processing and Regulation

Translation
-the synthesis of proteins as directed by mRNA templates. -carried out on ribosomes -Protein synthesis is thus the final stage of gene expression. involves interactions between three types of RNA molecules (mRNA templates, tRNAs, and rRNAs), as well as various proteins that are required for translation basic mechanics are also the same in all cells. polypeptide chains are synthesized from the amino to the carboxy terminus Each amino acid is specified by three bases (a codon) in the mRNA, according to a nearly universal genetic code.

1) mRNA transmits information present in DNA 2) tRNA - acts a bilingual translator molecule 3) Ribosomes and rRNA the workbenches for protein synthesis 4) tRNA synthetases - serve to mediate the attachment of specific amino acids to specific tRNA molecules. 5)amino acids

Transfer RNAs:
Transfer RNAs serve as adaptors that align amino acids on the mRNA template. Aminoacyl tRNA synthetases attach amino acids to the appropriate tRNAs, which then bind to mRNA codons by complementary base pairing.
Anticodon- The nucleotide sequence of transfer RNA that forms complementary base pairs with a codon sequence on messenger RNA. aminoacyl tRNA synthetase- An enzyme that joins a specific amino acid to a tRNA molecule carrying the correct anticodon sequence.

Activated form of amino acid

Anticodon

Figure 7.2. Attachment of amino acids to tRNAs

Common structural components:

1. The 3 terminal-CCA sequence to which the amino acid is bound. The encoded amino acid covalently attaches to the ribose of the terminal adenosine.
The other end of the molecule possesses a 3 nucleotide sequence called the anticodon which is the region of the molecule that attaches to the mRNA by complimentary base pairing.

2. A conserved cloverleaf secondary structure

3. An L-shaped three dimensional structure which allows them to properly fit into the ribosome.

* Each of the many molecular species has a unique specificity in interactions with mRNA and with the aminoacyl-tRNA synthetase that couples one specific amino acid to it.

All tRNAs share the same basic structure, since they all carry out the same basic function. However, they each possess a unique identifying sequence or anticodon. This is what allow them to interact with and incorporate the correct amino acid into the growing protein chain. - A molecule of tRNA contains 2 separate regions which allow it to act as a translator molecule between mRNA and proteins.
The incorporation of the correct amino acid into the protein is dependent on the proper amino acid attaching to the tRNA molecule and proper codon- anticodon base pairing. -One half of this is regulated by a group of enzymes called the Aminoacyl tRNA synthetases. - They serve to mediate the attachment of specific amino acids to specific tRNA molecules

Two-step activation of amino acids

The 3'-end adenine of a tRNA This ester bond gets readily hydrolyzed unless protected by a factor.

The Ribosome: particles composed of RNA and proteins that are the sites of protein synthesis.
The 4-letter language in mRNA is translated into 20-letter language in protein by ribosome.
Ribosomes in the cytoplasm Three binding sites of aa-tRNA across large and small subunits A passage for mRNA A tunnel for emerging peptide

Figure 7.4. Ribosome structure

Ribosomes on ER membrane

facilitate the coupling of the tRNA anticodons with mRNA codons. Each ribosome has a large and a small subunit formed in the nucleolus.

Ribosome is composed of proteins and ribosomal RNA (rRNA), the most abundant RNA in the cell. rRNA is transcribed in the nucleus, then bind to special proteins to form the ribosomal subunits in the nucleolus. The large and small subunits join to form a functional ribosome only when they attach to an mRNA molecule.

Eukaryotes encodes a single polypeptide (monocistric) the 5 end is capped (with 7 methylguanusine the 5- nontranslated region separates the cap from the translational initiation signal

Prokaryotes encodes more than one polypeptide (polycistronic) The 5 end is not capped initiation codons are preceded by the a specific nucleotide sequence called the Shine-Delgarno sequence.

Both prokaryotic and eukaryotic mRNAs have 5 and 3 non coding sequences. These are called the 5 UTR and 3 UTR.

* Two amino acids are designated by single codons: methionine by AUG and tryptophan by UGG

The rest are designated by two, three, four or six codons. Multiple codons for a single amino acid represent degeneracy in the code.
The genetic code is nearly universal. The same code words are used in all living organisms, prokaryotic and eukaryotic.

Non universal Codon Usage in Mammalian Mitochondria

Codon
UGA AUA AGA AGG

Usual Code
Termination Isoleucine Arginine

Mitochondrial Code
Tryptophan Methionine Termination Termination

Arginine

Translation of the codons of mRNA involves their direct interaction with complementary anticodon sequences in tRNA. Each tRNA species carries a unique amino acid, and each has a specific three-base anticodon sequence

codon-anticodon base pairing is Antiparallel codons are read in a sequential, nonoverlapping reading frame Anticodon and amino acid acceptor sites are located at opposite extremes of all tRNA molecules

Variances from standard base pairing are common in the codon-anticodon interactions. Many amino acids can be carried by more than one tRNA species, and degenerate codons can be read by more than one tRNA (but always one carrying the correct amino acid).

Wobble - the ability of one tRNA to recognize two or three different mRNA codons.
Occurs when the third base (5end) of the tRNA anticodon has some play or wobble, so that it can hydrogen bond with more than one kind of a base in the third position (3 end) of the codon.

E.g.: the base U in the wobble position of a tRNA anticodon can pair with either A or G in the third position of an mRNA codon

Some tRNAs contain a modified base called inosine (I), which is in the anticodons wobble position and can pair with U, C, or A in the third position of an mRNA codon. Thus, a single tRNA with the anticodon CCI will recognize three mRNA codons: GGU, GGC, or GGA all coding for glycine.

3 Codon Base A C G

5 Anticodon Bases Possible U or I G or I C or U

A or G or I

Some codons are read more effeciently by one anticodon than another. Not all codons are used equally, some being used very rarely. Examination of many mRNA sequnces has allowed construction of codon usage tables that show that different organisms preferentially use different codons to generate similar polypeptide sequences.

The Organization of mRNAs and the Initiation of Translation

UTR - untranslated regions

polycistronic -messenger RNAs that encode multiple polypeptide chains

monocistronic -messenger RNAs that encode a single polypeptide chain.

-both prokaryotic and eukaryotic cells, translation always initiates with the amino acid methionine, usually encoded by AUG. -In most bacteria, protein synthesis is initiated with a modified methionine residue (N-formylmethionine), whereas unmodified methionines initiate protein synthesis in eukaryotes (except in mitochondria and chloroplasts, whose ribosomes resemble those of bacteria).
Signals that identify initiation codons are different in prokaryotic and eukaryotic cells

Shine-Delgarno sequence -The sequence prior to the initiation site that correctly aligns bacterial mRNAs on ribosomes.

Figure 7.7. Signals for translation initiation

The Process of Translation

Figure 7.8. Overview of translation

Translation cycle

Only two sites are occupied at any time. Ribosome is a ribozyme. The crucial functions are mostly performed by rRNAs.

Table 7.1. Translation Factors Translation factors

Role

Prokaryotes

Eukaryotes

Initiation

IF-1, IF-2, IF-3

Elongation Termination

EF-Tu, EF-Ts, EF-G RF-1, RF-2, RF-3

eIF-1, eIF-1A, eIF-2, eIF-2B, eIF-3, eIF-4A, eIF-4B, eIF-4E, eIF-4G, eIF-5 eEF-1, eEF-1, eEF2 eRF-1, eRF-3

Initiation with initiation factors

This ester bond is labile and protected by eIF2.

In eukaryotes, small subunit binds to the cap of mRNA and moves to the first AUG codon. Eukaryotic mRNA are monocistronic due to this initiation mechanism. Some viral mRNA have an internal ribosome entry site (IRES) in the middle. In bacteria, small subunit binds to a Shine-Dalgarno sequence anywhere in mRNA to initiate translation. Thus, bacterial mRNA are polycistronic as they are produced from an operon.

Once an initiator codon has been recognize by the 40S subunit, the factors eIF1A and eIF3 bind to the 40s subunit and the eIF2 binds to the initiator methionyl tRNA.

eIF1 + initiator tRNA-eIF2 and 40s subuniteIF1a/eIF3

Pre initiator complex

the mRNA is recognized and brought into the ribosome by a group of factors associated with eIF4.

The 5 cap of mRNA is recognized by eIF4E which forms a complex with eIF4A and eIF4G. The eIF4G also bind to the poly A binding protein (PABP) which is associated with the poly A tail at the 3 end of mRNA.

It is the association of all of the eIF4 factors that serves to bring the mRNA to the 40S subunit..the eIF4G interacts with the eIF3 on the 40s subunit.

Then the 40S subunit with these factors and tRNA attached scans the mRNA for the initator (AUG) codon.

When the 40S ribosome complex encounters the AUG codon, the eIF5 initiates hydrolysis of the GTP bound to eIF2.

causes eIF2 which is bound to GTP to lose a phosphate. This means that eIF2 and any other associated factors are now only bound to GDP. This hydrolysis event, signals for the release of any factor associated with GDPthe removal of eIF2, eIF4A, 4, eIF1A, eIF1, eIF3, eIF4E and eIF4G This serves as the signal to allow the 60s Subunit to bind to the 40s Subunit. This is referred to as the 80s initiation complex.

Once this has formed, then elongation of the protein can begin.

Bacterial ribosomes engaged in elongating a polypeptide chain exist as 70S particles. Initiation of protein synthesis is not a function of intact ribosomes, but is undertaken by

the separate subunits, which reassociate during the initiation reaction.

Initiation occurs at a special sequence on mRNA called the ribosomebinding site. This is a short sequence of bases that precedes the coding region .The small and large subunits associate at the ribosome binding site to form an intact ribosome. The reaction occurs in two steps: Recognition of mRNA occurs when a small subunit binds to form an initiation complex at the ribosome-binding site. A large subunit then joins the complex to generate a complete ribosome.

Although the 30S subunit is involved in initiation, it is not by itself competent to undertake the reactions of binding mRNA and tRNA.
It requires additional proteins called initiation factors (IF). These factors are found only on 30S subunits, and they are released when the 30S subunits associate with 50S subunits to generate 70S ribosomes. This behavior distinguishes initiation factors from the structural proteins of the ribosome.

Several proteins called elongation factors take part in this three step cycle which adds amino acids one by one to the initial amino acid: 1. Codon recognition. 2. Peptide bond formation. 3. Translocation.

The ribosome as 3 sites for tRNA binding.the P or peptidyl site, the A or aminoacyl site and E or exit site. The A, E and P sites are used over and over during elongation.

The elongation cycle of translation - overview

(Prokaryotes)

1. Codon recognition

The mRNA codon in the A site of the ribosome forms hydrogen bonds with the anticodon of an entering tRNA carrying the next amino acid in the chain.
An elongation factor EF-Tu directs tRNA into the A site in bacteria. In eukaryotes eEF-1 (4 subunits: eEF-1, eEF-1, eEF-1, eEF-1) eEF-1 consists of eEF-11 and eEF-12 Hydrolysis of GTP provides energy for this step.

2. Peptide bond formation.

A peptide bond is formed between the polypeptide in the P site and the new amino acid in the A site by a peptidyl transferase. This reaction requires hydrolysis of GTP bound to EFTu, or eEF1. This inactivates EF-Tu, it is ejected from the ribosome and regenerated by EF-Ts. No eukaryotic homology of EF-Ts is known, but possibly one of the subunits of the eEF-1 has such activity. Peptidyl transferase activity appears to be one of the rRNAs in the large ribosomal subunit . The polypeptide separates from its tRNA and is transferred to the new amino acid carried by the tRNA in the A site.

3. Translocation.
The tRNA in the A site, which is now attached to the growing peptide, is translocated to the P site. Simultaneously, the tRNA that was in the P site is translocated to the E site and from there it exits the ribosome. During this process, the codon and anticodon remain bonded, so that mRNA and the tRNA move as a unit, bringing the next codon to be translated into the A site. The mRNA is moved through the ribosome only in the 5 to 3 direction. Translocation requires GTP hydrolysis and is mediated by EF-G in bacteria and by eEF-2 in eukaryotes.

3. Translocation.
The tRNA in the A site, which is now attached to the growing peptide, is translocated to the P site. Simultaneously, the tRNA that was in the P site is translocated to the E site and from there it exits the ribosome. During this process, the codon and anticodon remain bonded, so that mRNA and the tRNA move as a unit, bringing the next codon to be translated into the A site. The mRNA is moved through the ribosome only in the 5 to 3 direction.

Translocation requires GTP hydrolysis and is mediated by EFG in bacteria and by eEF-2 in eukaryotes.
This process continues until a termination codon is encountered by the ribosome.

Proofreading in elongation
Translation does not need to be as extremely accurate as DNA replication. Yet, there are several error correction steps in translation This ester bond is labile and protected by EF-Tu. All four base-pairs have identical Hbond patterns in the minor grooves. In all base-pairs, glycosidic bond positions are the same, which is used in checking correct base-pairings.

Termination of Translation

Typical stop codons are UAA, UAG, and UGA. When one of these sequences is translocated into the A site, this is the signal to stop translation. -Cells do not contain tRNAs with anticodons that are complimentary to these sequences, so it is impossible for translation to continue.

(Prokaryotes)

There are release factors that recognize this sequence and terminate protein synthesis. In eukaryotic cells, there is one release factor (eRF1) that recognizes all 3 stop codons.

Termination with release factors

The release factors resemble a tRNA to enter the A site and provides an H2O molecule to hydrolyze the last tRNA off the polypeptide Nascent peptide moves through a water-filled tunnel. The walls made of large subunit rRNA are like Teflon coating for easy sliding. Small hydrophobic spots are embedded in extensive hydrophilic surface of the wall. Bacterial mRNA are translated without processing before transcription termination. Eukaryotic mRNA form a circle to allow for rapid re-binding of ribosome.

What if you need lots of protein to carry out a particular function within a cell? -A single mRNA can be translated simultaneously by several ribosomes in both prokaryotic and eukaryotic cells. -Once a ribosome has moved away from the initaiton site another ribosome can bind and start synthesis of a new polypeptide chain. This is what you see here. -It is common for a single mRNA to be translated by several ribosomes spaces about 100-200 nucleotides apart. When this occurs and there are multiple ribosoem attached to the mRNA it is referred to as a polyribosome. Each ribosome synthesizes its own separate polypeptide chain. -This allows for a much more rapid syntheis of a given protein. Why might this be important? To allow a cell to respond to its environment quickly

Table 6-3. Inhibitors of Protein or RNA Synthesis

INHIBITOR SPECIFIC EFFECT Acting only on bacteria Tetracycline Streptomycin

blocks binding of aminoacyl-tRNA to A-site of ribosome prevents the transition from initiation complex to chain-elongating ribosome and also causes miscoding blocks the peptidyl transferase reaction on ribosomes (step 2) blocks the translocation reaction on ribosomes (step 3) blocks initiation of RNA chains by binding to RNA polymerase (prevents RNA synthesis) causes the premature release of nascent polypeptide chains by its addition to growing chain end binds to DNA and blocks the movement of RNA polymerase (prevents RNA synthesis) blocks the translocation reaction on ribosomes (step 3) blocks the peptidyl transferase reaction on ribosomes (step 2 ) blocks mRNA synthesis by binding preferentially to RNA polymerase II

Chloramphenicol Erythromycin Rifamycin

Acting on bacteria and eucaryotes Puromycin

Actinomycin D Acting on eucaryotes but not bacteria Cycloheximide Anisomycin -Amanitin

The ribosomes of eucaryotic mitochondria (and chloroplasts) often resemble those of bacteria in their sensitivity to inhibitors. Therefore, some of these antibiotics can have a deleterious effect on human mitochondria.