Translation 1

Translation

Vipin Shankar

The Central Dogma

Translation : overview Genetic information within the order of the nucleotides in mRNA is used to generate the linear sequences of amino acids in protein.

Overview… 







Most highly conserved event across all organisms. One of the most energetically costly event for the cell. More than 80% of the cell’s energy and 50% of dry weight is dedicated to protein synthesis in a rapidly growing cell. Synthesis of a single protein requires the coordinated action of over a 100 proteins and RNAs.

Challenge 





Translation is a more formidable challenge than transcription. Side chains of amino acids have little or no specific affinity to the nucleotide bases of mRNA. Direct interaction between mRNA template and amino acids could not be responsible for accurate ordering of amino acids in protein.

Adaptor molecule 



In 1955, Francis H Crick, proposed that prior to their incorporation into polypeptides, amino acids must attach to a special molecule that is capable of directly interacting with and recognizing the threenucleotide-long coding units of the mRNA. 2 years later Paul C Zamecnik and Mahlon B Hoagland discovered tRNA.

The Machinery     

mRNA. tRNA. Aminoacyl tRNA synthetase. Ribosome. Factors

Messenger RNA 







The translation machinery decodes only a portion of the mRNA. Information for protein synthesis is in the form of nucleotide triplets – Codons. The coding region is composed of contiguous, non-overlapping string of codons called Open Reading Frame (ORF). Each ORF specifies a single protein and starts and ends at internal sites within the mRNA.

Messenger RNA… 





Translation starts at the 5’ end of the ORF and proceeds one codon at a time to the 3’ end. The first and the last codons of an ORF are called the start & stop codons respectively. The start codon has 2 important functions: 



Specifies the amino acid to be incorporated into the growing polypeptide. Defines the reading frame for all subsequent codons.

Polycistronic & Monocistronic mRNA  





mRNAs contain at least one ORF. The number of ORFs vary in prokaryotes & eukaryotes. Prokaryotic mRNA usually contain 2 or more ORFs & hence encodes for multiple polypeptide chains – polycistronic mRNA. Eukaryotic mRNA generally have a single ORF – monocistronic mRNA.

The Ribosome Binding Site (RBS) 





 

For translation to occur, the ribosome must be recruited to the mRNA. Many pk. ORFs contain a short sequence typically located 3 -9 bps upstream of the start codon – the RBS, or the Shine- Dalgarno sequence. The RBS is complimentary to a sequence located near the 3’ end of one of the RNA components of the ribosome (16s rRNA). The core sequence is 5’ – CCUCCU – 3’ The extent of complementarity and the spacing between the RBS & the start codon influence the rate of translation.

Translational coupling 





In some pk. ORFs internal to a polycistronic message lack a strong RBS, but are nonetheless actively translated. In these cases the start codon often overlaps the 3’ end of the adjacent ORF (5’ –AUGA- 3’) A ribosome that has just completed translation of the upstream ORF is appropriately positioned to begin translating from the start codon of the downstream ORF, circumventing the need for fresh ribosome recruitment.

Ek. mRNAs are modified 







Ek. mRNAs recruit ribosomes using a chemical modification called the 5’-cap. The 5’ cap is a methylated guanine nucleotide that is joined to the 5’ end of the mRNA through a 5’-5’ linkage. The resulting structure recruits the ribosome to the mRNA. Once bound the ribosome moves in a 5’-3’ direction until it encounters a start codon – Scanning.

Ek. mRNA… 





In some mRNAs, a purine base is present 3 bases upstream of the start codon and a ‘G’ immediately downstream. (5’-G/ANNAUGG- 3’) – Kozak sequence. Its presence increases the efficiency of translation. This sequence interact with initiator tRNA.

Ek. mRNA… 





At the extreme 3’ end of the mRNA a chain of Adenine nts are present – the poly A tail. This tail is added enzymatically by the enzyme poly-A polymerase. Despite its location at the 3’ end, the poly-A tail enhances the rate of translation, by promoting efficient recycling of ribosomes.

Transfer RNA 





tRNAs are adaptors between codons and amino acids. There are many types of tRNAs, but each is attached to a specific amino acid and each recognizes a particular codon, or codons. All though the exact sequence varies, all tRNAs have certain features in common.

Common features of tRNAs 







All tRNAs end at 3’ end with the sequence 5’ – CCA- 3’ This is the site that is attached to the cognate amino acid. Several unusual bases are present in the tRNAs’ primary structure. The unusual bases are created posttranscriptionally, by enzymatic modifications of the normal bases.

Attachment of amino acid to tRNA  





Charging of tRNA. Acyl linkage between, the carboxyl group of the amino acid and the 2’ or 3’ hydroxyl group of the adenosine nucleotide that protrudes from the acceptor stem. This is high-energy bond, and the hydrolysis of this bond results in large change of free energy. The energy released when the bond is broken drive the formation of peptide bond during translation.

Charging… 

Aminoacyl tRNA synthetase, attach an amino acid to a tRNA in a 2 step reaction. 



Step 1: Adenylylation: amino acid reacts with ATP to become adenylylated with the concomitant release of pyrophosphate. Adenylylation refers to the transfer of AMP, opposed to adenylation which indicates the transfer of adenine.

Charging…

PPi

Charging… 



As a result of adenylylation, the amino acid is attached to adenylic acid via a highenergy ester bond in which the carbonyl group of the amino acid is joined to the phosphoryl group of AMP. The adenylylated amino acid remain tightly bound to the synthetase.

Charging… 



Step 2: adenylylated amino acid is transferred from the enzyme to the 3’ end of the tRNA via the 2’- or 3’ – hydroxyl and the concomitant release of AMP. There are 2 classes of tRNA synthetases. 



Class I enzymes attach amino acids to 2’ OH of tRNA and are generally monomeric. Class II enzymes attach amino acids to 3’ OH of tRNA and are typically dimeric or trimeric.

General Structure of amino acyl tRNA

Charging… 





Each of the 20 amino acids is attached to the appropriate tRNA by a single, dedicated tRNA synthetase. Because, most amino acids are specified by more than one codon, it is not uncommon for one synthetase to recognize and charge more than one tRNA: Isoaccepting tRNAs. Nevertheless, the same tRNA synthetase is responsible for charging all tRNAs of a particular amino acid.

Specificity of charging 

tRNA synthetases: 



Must recognize the correct set of tRNAs for a particular amino acid, (specificity of tRNA recognition) and must charge all these isoaccepting tRNAs with the same amino acid (Accuracy of amino acyl tRNA formation).

Specificity of tRNA recognition 



tRNA synthetases recognize some specific regions of the tRNA which help them to identify the correct cognate tRNA – specificity determinants. Specificity determinants are clustered at 2 distinct sites on the tRNA 

the acceptor stem - discriminator. the anti-codon loop – anti-codon.

Specificity of tRNA recognition… 







The anti-codon dictates the amino acid, that the tRNA is responsible for incorporating. However, because each amino acid is usually specified by more than one codon, recognition of anti-codon cannot be used in all cases. So the discriminator plays a greater role in tRNA recognition. The set of tRNA determinants that enable synthetases to discriminate among tRNAs is referred to as ‘the second genetic code’.

Accuracy of amino acyl tRNA formation 





The challenge to recognize the correct amino acid is even more daunting. The relatively small sizes and the similarity makes the task difficult. Despite this challenge, the frequency of mischarging is very low: typically less than 1 in 1000 tRNAs is charged with incorrect amino acid.

Accuracy of amino acyl tRNA formation... 



Different synthetases use different mechanisms to distinguish between the amino acids. Eg 1. 

Tyrosyl tRNA synthetase: the oppertunity of forming a strong and energitically favourable hydrogen bond with the hydroxyl moiety of tyrosine helps the synthetase to identify tyrosine.

Accuracy of amino acyl tRNA formation... 

Eg 2. 





Isoleucine and valine differ only by a single methyl group. Valyl tRNA synthetase can sterically exclude isoleucine from its catalytic pocket as isoleucine is larger than valine.

Eg 3. 



But valine can easily slip into the isolucyl tRNA synthetase catalytic pocket. The interaction of the methyl group on isoleucine gives an extra -2 to -3 Kcal/mol of free energy. This relatively small energy difference makes the reaction 100 times more likely.

Accuracy of amino acyl tRNA formation... 







One common mechanism to increase fidility of an amino acyl tRNA synthetase is to proofread the products of the charging reaction. This is performed by an editing pocket present in addition to the catalytic product. All wrongly charged amino acyl tRNAs enter the editing pocket, while the correctly charged amino acyl tRNA is sterically excluded (molecular sieve). Within the editing pocket the amino acyl tRNA is hydrolyzed.

Accuracy of amino acyl tRNA formation... 



The ribosome is unable to discriminate between the correctly and incorrectly charged tRNAs. The ribosome ‘blindly’ accepts any tRNA that exhibits a proper codon – anti codon interaction, whether or not the tRNA carries its cognate amino acid.

The Ribosome 







A macromolecular machine that directs the synthesis of proteins. Ribosome is larger and more complex than the minimal machinery required for DNA and RNA synthesis. Ribosome is composed of at least 3 different RNAs and 50 different proteins with an overall molecular mass of over 2.5 mega daltons. The rate of translation is very slow (2-20 aa/sec).

Ribosome…  





Composed of a large and a small subunit. Large subunit contains the peptidyl transfer center, responsible for the formation of the peptide bond. The small subunit contains the decoding center, in which the charged tRNAs read or ‘decode’ the codon units of the mRNA. The large and small subunits are named according to the velocity of their sedimentation when subjected to a centrifugal force (Svedberg unit).

The ribosome cycle 

Central to the mechanism of translation is a cycle in which the small and the large subunits of the ribosome associate with each other and the mRNA, translate the target mRNA, then dissociate after each round of protein synthesis: ribosome cycle.

The polysome 



Although a ribosome can synthesize only one polypeptide at a time, each mRNA can be translated simultaneously by multiple ribosomes. An mRNA containing multiple ribosomes is called polyribosome or polysome.

tRNA binding sites 

  

Ribosome has 3 tRNA binding sites called A, P and E sites. A site for amino acyl tRNA. P site for peptedyl tRNA. E site is the exit site.

The process of translation 

3 steps   

Initiation. Elongation. Termination.

Initiation of translation 

3 events  





Recruitment of ribosome. Placement of charged tRNA into the P site of the ribosome. Precise positioning of the ribosome over the start codon.

The positioning of the ribosome over the start codon is critical since it establishes the reading frame for the translation of mRNA.

Initiator tRNA.  







First charged tRNA to enter the ribosome. Base pairs with the start codon AUG or GUG. AUG and GUG have different meanings within the ORF (met & val). Initiator tRNA is charged with N-formyl methionin (fmet). Initiator tRNA depicted as fMet-tRNAifMet

Initiation factors  





IF1, IF2 & IF3 IF1- prevents initiator tRNA from binding to A site. IF2 – a GTPase interacts with the small subunit, IF1 and initiator tRNA. Facilitates subsequent association of initiator tRNA with the small subunit and prevents other charged tRNAs from binding. IF3 – binds with small subunit and prevents its from associating with the large subunit.

Initiation in prokaryotes 







Step 1: IF3 binds with small subunit of ribosome near the E site. Step 2: IF1binds the small subunit of ribosome near the A site. Step 3: IF2 in association with a GTP molecule bids to IF1. Step 4: The mRNA and the fMet-tRNAifMet associate with the assembly.

Initiation in prokaryotes… 





Step 5: Ribosome positioning by complimentary pairing of 16s rRNA and the Shine Dalgarno Sequence. This complex is called the 30s initiation complex. Step 6: IF3 falls off. Large subunit associates with small subunit.

Initiation in prokaryotes… 





Step 7: hydrolysis of GTP by IF2 is initiated by the factor binding center of the large subunit. IF2 – GDP has less affinity to ribosome hence IF2, GDP and IF1 are released. This is the 70s initiation complex.

mRNA

70s initiation complex

30s initiation complex

Initiation in eukaryotes    

Similar to that in prokaryotes. More number of initiation factors present. A little more complex than in prokaryotes. Initiator tRNA is charged with methionine (met).

Initiation in eukaryotes… 











Step 1: factors eIF6, eIF3 & eIF1A (analogous to IF3 & IF1 in pk), bind to ribosome- ribosome dissociates into small and large subunits. Step 2 : eIF2 – GTP forms complex with initiator tRNA [eIF2-GTP- Met-tRNAiMet]. Step 3: eIF5B – GTP associates with small subunit of ribosome in a eIF1A dependent manner. Step 4: eIF5B helps to recruit [eIF2-GTP- Met-tRNAiMet] complex to the small subunit. The initiator tRNA is positioned in the P site of the small subunit. This complex is called the 43S-pre-initiation complex.

Initiation in eukaryotes… 



Step 5: eIF4E subunit of eIF4 binds to the 5’ cap of mRNA. During the same step, 2 other subunits of eIF4 binds with the mRNA non-specifically – forming mRNA-eIF4 complex. Step 6: eIF4B activates helicase activity in another subunit of eIF4. 



The helicase unwinds any secondary structures that may have formed in the mRNA. Removal of secondary structures is critical for the binding of the small subunit of the ribosome.

Initiation in eukaryotes… 



Step 7: The mRNA – eIF4 complex associates with the 43s pre-initiation complex through the interaction between eIF4F and eIF3. This is now called the initiation complex.

Initiation in eukaryotes… 

Step 8: The initiation complex move along the mRNA in a 5’-3’ direction in an ATP dependent process, driven by the eIF4 associated RNA helicase. 





During this movement, the small subunit scans the mRNA for the first start codon. The start codon is recognized by the base pairing between the anti codon of the initiator tRNA and the start codon. Kozak sequence points out the correct start codon.

Initiation in eukaryotes… 





 

Step 9: Correct base pairing triggers the release of eIF2 and eIF3. Step 10: The large subunit attaches to the small subunit. Step 11: The remaining factors are released by stimulating GTP hydrolysis by eIF5B. Step 12: The initiator tRNA is placed in the P site. This is the 80s initiation complex.

Exceptions to normal initiation 

uORFs 









Not all ek polypeptides are encoded by an ORF that starts with the AUG that is the most proximal to the 5’ terminus. In some cases, the first AUG is not a proper sequence context, resulting in its bypass. In other cases, short, upstream ORF (uORF) encoding peptides less than 10 aa, are found upstream to the principal ORF. The uORF act to regulate the rate of translation of the downstream ORF. The uORF is followed by a sequence of RNA that retains the initiation complex, which continue scanning of the mRNA for AUG

Exceptions to normal initiation 

IRES 





Internal Ribosome Entry Sites (IRES) are RNA sequences that function like the pk ribosome binding sites. They recruit the small subunit to bind and initiate at an internal site in the mRNA. This method is also exhibited by viral mRNAs.

Translation initiation factors hold ek mRNA in circles. 





In addition to binding the 5’ end of ek mRNA, the initiation factors are closely associated with the 3’ end of the mRNA through its poly-A tail. This is mediated by an interaction between eIF4F and Poly-A binding protein that coats the poly-A tail. These interactions hold the mRNA in a circular configuration via a protein bridge between the 5’ and 3’ ends of the molecule.