9 mRNA and its role in protein biosynthesis

Prof. Sunil Kumar Khare

  1. Objectives
  • What is mRNA
  • Difference between prokaryotic and eukaryotic mRNA and protein synthesis Structure of mRNA
  • Role of 7’ methyl guanosine cap, 5’ UTR, 3’ UTR and poly A tail in protein synthesis

 

What is mRNA

 

Messenger RNA or mRNA transfers information from DNA to synthesize protein with the help of rRNA and tRNA.

 

 

Difference between prokaryotic and eukaryotic mRNA and protein synthesis

 

Eukaryotic RNA is monocystronic contains codons of a single cistron that codes only for single protein and has one initiation and termination codon. While prokaryotic are polycyctronic contains codons for more than one cistron and codes more than one protein. They have more than one gene and has many initiation and termination codons (Fig 1).

 

Fig 1: Prokaryotic and Eukaryotic mRNA

The mechanism of protein initiation and engage of mRNA, ribosome differ in prokaryotes and eukaryotes. Initiation site in polycistronic prokaryotes usually done by base paring with rRNA. Contrary to this, in eukaryotes mRNA reached to initiation site by scanning mechanism that starts from AUG codon present near the 5’ end of mRNA.

 

Selection of start site for protein synthesis in prokaryotic and eukaryotic mRNA

 

Prokaryotes

 

In the upstream of prokaryotic mRNAs a purine-rich (ACCUCCUUA) sequence called Shine–Dalgarno (SD) sequence is present that make pairing with complementary sequence present in the smaller ribosomal subunit of 16S rRNA. This complex was first time evident by Steitz and Jakes in 1975 when they isolate a complex between 30 nt fragment of mRNA and 3’ fragment of 16S rRNA from coliphage R17. The sequence consists of 3-9 adjoining bases in the mRNA which pairs with some or all bases at the 3’ end of 16S rRNA. The spacing between the SD sequence and the initiation codon varies with the average being 7 nucleotides (nt) and this distance determines the efficiency of translation. The optimal spacing between the SD and the AUG initiation codon is 5 to 13 nt (Fig 2).

A) Ribosomal binding site having Shine Dalgarno sequence and the initiation codon AUG. The shine dalgarno sequence is complementary to the 3’ end of 16S rRNA and the anti -codon of fMet-tRNA is also shown in the figure.

B) Ribosomal binding site with partial shine dalgarno sequence, here SD-AUG spacing is defined as the number of nucleotide separating the partial SD and AUG.

C) The ribosomal binding site with a partial shine dalgarno sequence. The SD-AUG spacing and the aligned spacing are the same.

 

There is a requirement of at least three base pairs for SD interaction with 16S it could be with AUG or with a weaker codon like UUG or GUG. However, non-AUG codon that lacks SD sequence can start up very low level of translation initiation by coupled translation. This can be consummated by upstream cistron that finished near to the UUG or GUG codon of the next cistron that brings ribosomes to the internal start site.

The SD interaction apparently promotes the anchoring of 30S subunit in the close proximity of the start codon. The weak interaction of ribosomes and SD sequence when bounded with the non-AUG codon could be detected with the help of toe printing assay. When mRNA is in unfolded stage, the SD

 

interaction has ability to reshuffle the secondary structure which hampers access to the AUG codon. Many researchers have shown that even after formation of the first peptide bond both mRNA and SD sequence remains in paired form. It is still not known at what point this contact is broken. It is interesting to know that during elongation the anti-SD sequence present in 16S rRNA stabilize ribosome/mRNA complexes. In most of the bacterial mRNAs, initiation depends on the accessibility of SD sequence and nearby AUG codon to the ribosome.

 

Working of Polycistronic mRNAs in prokaryotes

 

In polycistronic mRNA translation initiation can occur at multiple sites and translation may be affected by the neighboring cistrons. However, translation initiation first takes place with that cistron which has stronger ribosomal binding affinity. This is often regulated by the SD/AUG bonding and a part of upcoming coding sequence. The base pairing is interrupted when ribosome advanced in the upcoming cistron as a result downstream cistron gets activated. Thus, elongating ribosome disrupt secondary structure which is inhibitory to the upcoming ribosome. Translation coupling is the important mechanism that coordinates the synthesis of ribosomal protein. In some cases same ribosomal subunits are used for the upcoming cistron this mechanism is called reinitiation. There is a vast difference between reinitiation in prokaryotes and eukaryotes. In prokaryotes there is no limitation on the size of upstream cistron, also the end of first cistron frequently overlies with the start of second for eg (  ). The similar arrangement is not possible in eukaryotes. Few studies on E. coli showed that after subsequent termination the loosened ribosome may move toward the rear or back to locate reinitiation site.

 

Eukaryotes

 

Translation initiation in eukaryotic mRNA

 

It is totally different from the prokaryotic initiation mechanism. To begin the eukaryotic translation mechanism smaller ribosomal subunit (40S) having Met-tRNAi.eIF2.GTP first enter to the 5’ end rather than at AUG codon. Then they migrate through 5’ UTR till they reached to AUG codon to take a pause where 60S ribosomal subunit joins with 40S and fix the selection of start codon. In eukaryotic there is no alternative initiation codon in place of AUG unlike prokaryotes that may initiate at GUG or UUG. In eukaryotes neighboring sequences determines the efficiency by which the first AUG codon is acknowledged and act as a small pause during scanning phase of initiation. Vertebrate mRNAs, traditionally have consensus sequence flanking AUG is “GCCRCCaugG”. Within this consensus sequence, purine (R) positioned at -3 nt from first A of AUG is highly conserved, usually at position A-3 is found in most of the cases. Many researcher has reported that mutation at this position strongly impair initiation both invitro and invivo. Similarly, a study confirms an important role of G+4 in the initiation step. There is a marginal contribution of other consensus sequences if an AUG codon is flanked by A−3, or by G−3 and G+4. Thus, AUG codon can be a weak or strong start site based on the presence of nucleotide at position -3 and +4. However how these consensus sequence are recognized and how they function is still not know, but it was predicted that it works by scanning mechanism. Many researchers have proposed the scanning mechanism for eukaryotic mRNA but only four experimental mechanisms supports the hypothesis that 40S enters at 5’ end of the mRNA. These are as follows:

  • Eukaryotic ribosomes are could not bind to the synthetic circular mRNA.
  • Translation of most of the mRNA depends upon 5’ m7G cap.
  • Abortive initiation was noticed in β-globin mRNA having trimmed 5’ end.
  • Ribosome binding was inhibited when repressor blocks 5’ end.

There are few other experiments which show that after making entry at 5’ end ribosome migrate through mRNA (scanning mechanism). These are as follows:

  • After binding to mRNA the 40S subunit can breach some base pair until it sensed the stable base pairing as confirmed by toeprinting experiments.
  • The antibiotic edeine that prejudice the recognition of AUG ‘stop codon’, allows 40S subunits to pack the entire length of the mRNA.
  • Besides this, the translation fails completely when there is a formation of stable stem-and loop structure that blocks access to the first AUG codon, which also supports scanning mechanism.

It is interesting to mention here AUG codon could be recognized better when a small hairpin loop was introduced between 13 to 15 nt towards downstream of AUG as confirmed by mapping experiments. A hairpin structure supposed to pause 40S ribosomal subunit with its AUG-recognition-center right over the AUG codon.

 

Sometimes eukaryotic mRNA undergoes leaky scanning, which means that initiation starts at second or third AUG codon instead of first.

 

Leaky scanning

 

Leaky scanning occurs due to dearth of fine context near the first AUG codon (Fig. 3, line 3). Almost two dozen mRNAs were found to have 5’ proximal AUG codon present in a suboptimal background, thus ribosomes initiate both at first and second AUG codons, producing two variable proteins from one mRNA. Most of the viruses, including HIV, have employed leaky scanning to make essential proteins undergoes the importance of context (A−3, G+4). The extent of leaky scanning may vary depending upon the neighboring nucleotides of first AUG and downstream secondary structures. Even initiation from the second AUG codon can be repressed when row of elongating ribosomes, moving forward from the upstream start site, prevents access to the downstream site. This occlusion can be reduced by moving second start site nearer to the first. Beside this, the first AUG codon can also be bypassed if it is too close to the 5’ end to be recognized efficiently than eIF-2. Leaky scanning may also occurs when initiation starts at a non-AUG codon, such as ACG, CUG or GUG. However, in higher eukaryotes, pairing among Met-tRNA and non-AUG codons is weak, only alternative codons that have good base pairing (bonding) are functional in mammals. While leaky scanning is exercised regularly as measures to make two functional proteins, intermittently an upstream initiation site exists only for regulatory purposes. Recent experiments recommended that leaky scanning can be amended by temperature, growth phase. Let’s see how reinitiation works in eukarotes.

Dashed lines show the pathway for 40S subunit. The limit of initiation to the first AUG codon (line2) can be avoided by reinitiation (line 2) or context reliant leaky scanning (line 3).

 

Reinitiation

 

In eukaryotes reinitiation starts after translation of the first small ORF (upORF). When ribosome reached to a termination codon the 40S subunit can stick on the mRNA to restart scanning and reinitiate at downstream AUG codon (Fig. 3, line 2). The size of the upORF contributes to the ability of eukaryotic ribosomes to reinitiate the protein synthesis. A study suggested that the cut- off length of upORF was about 30 codons. This means that one mRNA can make one small peptide and one full-length protein but not two complete proteins. It is most efficient when upORF end few nucleotides prior to the start site of the upcoming cistron as 40S subunit requires time to regain Met-tRNA· eIF-2 for the acknowledgment of downstream AUG codon. Eukaryotes could not use reinitiation to produce two full-length proteins, therefore, its main purpose is something different. In some cases, translation is synchronized in complicated manners by assets of the upORF or by the small peptide encoded by the same mRNA. It is incompetent when upORFs are engaged in lowering the translational efficiency. In some viruses, mRNA should be free of ribosomes in order to replicate and package and this phenomenon is promoted by upORFs. Similar case was observed in retroviruses and picornaviruses. In cellular mRNAs, upORFs are useful in limiting the translation of some proteins which are toxic (toxic to the cell) when over produce. When the former protein is required by the cell these inhibitory upORFs may be eliminated by splice site. One such example is hereditary thrombocythaemia where splicing mutation eradicates upORFs and thus constitutively boosts the development of thrombopoietin.

 

Eukaryotic mRNA is itself has very complicated structure and has several regions monitoring various functions during translation.

 

Structure of eukaryotic mRNA

 

Eukaryotic mRNA from 5’ to 3’ end consists of 7 methyl guanosin cap, 5’ un transcribe region (UTR), exone, 3’ UTR and poly A tail (Fig 4).

 

Figure 4: structure of eukaryotic mRNA

7 methyl guanosin cap

 

The cap present at 5’ end of mRNA is known to protect the mRNA against degradation by cellular nucleases. Beside this it also helps in initiation of protein synthesis and binding of riboso mes to the mRNA. Sequence studies were conducted on reovirus mRNA which showed that it contains some site which was recognized by ribosome and help in their attachment. Methylation of the terminal guanosine residue is of prime importance as it facilitates translation and in some cases eIF-4E arbitrates the cap dependent step in initiation. The dependence of leader sequences on the m7G cap is evocative of the circumstances in prokaryotes in which secondary structure near to the initiation domain enhances dependence on the SD sequence. In both cases, it can be explained by the fact that it might have an extra point of connection with eIF-4E in eukaryotes or the SD sequence in prokaryotes stabilizes the ribosome/mRNA interaction until the adjacent mRNA segment breathes. In eukaryotes, in vivo translation initiation is totally dependent on m7G cap. However, artificial conditions can be generated to translate unmethylated mRNA by adding the 5’ UTR from a picornavirus. In addition transcripts synthesized by RNA polymerase III are cap independent and less than 1% transcripts undergoes translation at very low frequency. An important theoretical point is that, even when conditions allow cap independent translation, ribosome binding remains 5’ end-dependent. However, on comparing translation of methylated and unmethylated mRNAs of vaccinia virus Weber et al established that role of 7- methylguanosine cap for translation was strongly influenced by K+ concentration. If the concentration of K+ ion is low then 7 methyl guanosine cap doesn’t play much efficient role the rate of translation rate in methylated and un-methylate mRNA were same. On the other hand in vivo where K+ ion concentration is high (150-160 mM), protein initiation was facilitated by the presence of 7 methyl guanosine cap. Under these conditions translation was reduced to 70-80 % in decapped mRNAs.

 

5’ UTR (Untranslated region)

 

Structure of 5’ UTR plays important role in the initiation and protein translation. Researchers have independently done many experiments to understand the role of 5’UTR. In one experiment it was found that introduction of stable stem-loop structure with energy of -30kcal/mol, placed 12nt away from m7G cap can profoundly diminished translation. However, when same structure was introduced around 52 nt downstream of m7G cap it did not affect the translation. This is because when stem loop is near to the cap it blocks the access of 43S initiation complex (assembly of 40S plus 3 initiation factors) to the mRNA. When distance between stem loop is large that 43S complex get enough time to melt the stem loop structure and continue the translation without any hindrance. In contrast when more stable hairpin loop having energy of -61 kcal/mol was introduced into 5’ UTR it could suppress initiation even after placing it to 71 nt downstream of m7G cap. Thus, this implies that inhibitory effect of secondary structure in UTR region depends on the stability as well as position of the hairpin. Although these hairpins loop adversely affect the translation, but for the selection of AUG codon it acts positively. In mRNA which lacks purin at -3 or a G at +4 position the 43S complex ignores it and continu further downstream for the scanning of next AUG codon (leaky scanning).

 

3’ UTR and poly A tail

 

Recently it was detected that the interactions of poly(A) binding protein (PABP) and certain initiation factors has aroused a where the 3’ end of the mRNA assembles and brings initiation complexes to the 5’ end. The poly(A) tail and associated PABP has ability to promote translation by avoiding the diffusion of initiation factors or, via interactions with the cytoskeleton, avoiding diffusion of the mRNA.