Mechanism of mRNA binding to bovine mitochondrial ribosomes

The binding of mRNA to bovine mitochondrial ribosomes was investigated using triplet codons, homopolymers and heteropolymers of various lengths, and human mitochondrial mRNAs. In the absence of initiation factors and initiator tRNA, mitochondrial ribosomes do not bind triplet codons (AUG and UUU) or homopolymers (oligo(U] shorter than about 10 nucleotides. The RNA binding domain on the 28 S mitoribosomal subunit spans approximately 80 nucleotides of the mRNA, judging from the size of the fragments of poly(U,G) and natural mRNAs protected from RNase T1 digestion by this subunit, but the major binding interaction with the ribosome appears to occur over a 30-nucleotide stretch. Human mitochondrial mRNAs coding for subunits II and III of cytochrome c oxidase and subunit 1 of the NADH-ubiquinone oxidoreductase (complex I) were used in studying in detail the binding of mRNA to the small subunit of bovine mitochondrial ribosomes. We have determined that these mRNAs have considerable secondary structure in their 5'-terminal regions and that the initiation codon of each mRNA is sequestered in a stem structure. Little mRNA was bound to ribosomes in a manner conferring protection of the 5' termini from RNase T1 digestion, under standard conditions supporting the binding of artificial templates, but such binding was greatly stimulated by the addition of a mitochondrial extract. Initiation factors and tRNAs from Escherichia coli were unable to stimulate the 5' terminus protected binding of these mRNA molecules, demonstrating a requirement for homologous factors. Our results strongly suggest that mitochondrial initiation factors are required for the proper recognition and melting of the secondary structure in the 5'-terminal region of mitochondrial mRNAs, as a prerequisite for initiation of protein synthesis in mammalian mitochondria.     

Ribosome binding site analysis of ovalbumin messenger ribonucleic acid.

The region of the ovalbumin messenger ribonucleic acid (mRNAov) molecule bound to the 40S ribosomal subunit and its associated initiation factors in the wheat germ cell-free translation system were isolated and characterized. Two mRNAov fragments, 87 and 92 nucleotides in length, were protected from T1 ribonuclease digestion by binding of guanosine 5',beta,gamma-methylenetriphosphate and were shown by hybridization and fingerprint mapping to be derived from the 5' end of mRNAov. Both these mRNAov fragments were of sufficient length to contain both the cap structure and the AUG initiation codon. Four T1-resistant oligonucleotides, prepared by direct digestion of mRNAov with T1 ribonuclease were also found to bind to the wheat germ 40S ribosomal subunit. Nucleotide sequence analysis of these oligonucleotides revealed (1) that they were not a subset of the ribosome binding fragments described above, (2) that they were derived from within the mRNAov molecule (one from within the coding region and three from the noncoding region at the 3' end of the mRNAov molecule), and (3) that three of the four mRNAov nucleotides contained 3'-terminal AUG trinucleotides. These data suggested that features of the mRNAov molecule in addition to the nucleotide sequence might be important in specifying the correct ribosome binding site for the initiation of protein synthesis. The amount of mRNAov bound to the wheat germ 40S ribosomal subunit in a preinitiation complex was found to vary inversely with the potassium ion concentration. Lowering the potassium concentration to levels suboptimal for translation also resulted in the protection of larger fragments of the mRNAov molecule derived from the same 5'-end region as the ribosome binding fragments described above. The ability of the cap analogue 7-methylguanosine 5'-phosphate (m7G5'p) to reduce the amount of mRNAov bound to the wheat germ 40S ribosomal subunit was found to depend directly on thepotassium concentration. Interestingly, the effects of potassium on the amount of mRNAov bound in a preinitiation complex and the inhibition of this binding by m7G5'p could be observed by changing the potassium concentration after binding had occurred. These data suggested that the interaction between the wheat germ 40S ribosomal subunit and mRNAov was very sensitive to the ionic environment.     

Complementarity between ferritin H mRNA and 28 S ribosomal RNA

We have found an interesting complementarity in sequences of human ferritin H mRNA and 28 S ribosomal RNA. Immediately upstream of the initiating AUG in the ferritin mRNA is a stretch of 67 nucleotides which contains sequences complementary to several regions in 28 S RNA. One such region can form 55 base pairings with the 5' noncoding region of the ferritin H mRNA. Most of the complementarity is due to repeats of CCG in the ferritin mRNA and GGC in the ribosomal RNA. The regions of complementarity in the 28 S RNA appear to be expansion sequences that have arisen in the evolution of eukaryotic ribosomal RNA. We suggest that interaction of ferritin mRNA and 28 S RNA may function to regulate the stability and/or translatability of ferritin mRNA.     

Identification of features in 5' terminal fragments from reovirus mRNA which are important for ribosome binding

Four types of experiments were carried out with reovirus messenger RNAs or with 5′ terminal fragments of known sequence to identify features in mRNA which appear to be important for formation of initiation complexes with ribosomes. With a number of reovirus mRNAs, 40S initiation complexes had been previously shown to protect a significantly larger segment of the RNA (including the 5′ terminal m7G) than that protected by 80S initiation complexes. Each 80S-protected sequence had an AUG codon and was a subset of the 40S-protected sequence from the same message. When 40S- and 80S-protected fragments were tested for ability to rebind to ribosomes, the 80S-protected fragments showed considerably lower binding ability, implying that the “extra” sequences protected by 40S initiation complexes contribute to ribosome attachment. Nevertheless, wheat germ ribosomes select the same 5′ terminal initiation site in each reovirus mRNA, irrespective of the presence or absence of m7G on the message. This was demonstrated by comparing fingerprints of the ribosome-protected regions obtained with methylated versus unmethylated RNA. The contribution of m7G to formation of initiation complexes is therefore quantitative rather than qualitative. Limited T1 RNAase digestion of isolated 5′ terminal fragments from several reovirus messages generated a series of smaller fragments which were analyzed for ability to rebind to ribosomes. Partial digestion products up to 30 nucleotides in length which retained the 5′ cap but not the AUG codon were unable to associate stably with ribosomes, whereas every AUG-containing fragment that was analyzed was able to form initiation complexes. The efficiency of binding of certain AUG-containing fragments, however, was reduced by removal of either the 5′ terminal region, including the cap, or of sequences comprising the beginning of the coding region, on the 3′ side of the AUG. Complex formation between messenger RNA and ribosomes was inhibited by the trinucleotide AUG, but not by various other oligonucleotides. Although the inhibition was specific, a vast excess of trinucleotide was required for moderate inhibition of 80S complex formation, and the same concentration of AUG failed to inhibit formation of 40S initiation complexes.     

Domains on the hepatitis C virus internal ribosome entry site for 40s subunit binding

The internal ribosome entry site (IRES) of the hepatitis C virus (HCV) RNA is known to interact with the 40S ribosomal subunit alone, in the absence of any additional initiation factors or Met-tRNAi. Previous work from this laboratory on the 80S and 48S ribosomal initiation complexes involving the HCV IRES showed that stem-loop III, the pseudoknot domain, and some coding sequence were protected from pancreatic RNase digestion. Stem-loop II is never protected by these complexes. Furthermore, there is no prior evidence reported showing extensive direct binding of stem-loop II to ribosomes or subunits. Using direct analysis of RNase-protected HCV IRES domains bound to 40S ribosomal subunits, we have determined that stem-loops II and III and the pseudoknot of the HCV IRES are involved in this initial binding step. The start AUG codon is only minimally protected. The HCV-40S subunit binary complex thus involves recognition and binding of stem-loop II, revealing its role in the first step of a multistep initiation process that may also involve rearrangement of the bound IRES RNA as it progresses.     

Feedback inhibition of poly(A)-binding protein mRNA translation. A possible mechanism of translation arrest by stalled 40 S ribosomal subunits

An adenine-rich cis element at the 5'-untranslated region (UTR) of Pabp1 mRNA is able to inhibit translation of its own mRNA. Similar inhibition of translation of a reporter beta-galactosidase mRNA is observed when the adenine-rich auto regulatory sequence (ARS) is placed within the 5'-UTR of this mRNA. For this translational control the distance of the ARS from the 5' cap is not important. However, it determines the number of 40 S ribosomal subunits bound to the translationally arrested mRNA. Inhibition of mRNA translation by this regulatory sequence occurs at the step of joining of the 60 S ribosomal subunit to the pre-initiation complex. Translational arrest of the ARS containing mRNA in a rabbit reticulocyte lysate cell-free system in the presence of exogenous Pabp1 protects the 5'-flanking region of the ARS from nuclease digestion. This protection depends on the binding of the 40 S ribosomal subunit to the mRNA. The size and the sequence of the nucleotide-protected fragment depends on the location of the ARS within the 5'-UTR. When the ARS is located at a distance of about 78 nucleotides from the 5' cap, a 40-nucleotide long region adjacent to the ARS is protected. On the other hand, when the ARS is moved further away from the 5' cap to a distance of approximately 267 nucleotides, a 100-nucleotide-long region adjacent to the ARS is protected from nuclease digestion. Nuclease protection is attributed to the presence of one or more stalled 40 S ribosomal subunits near the Pabp1-bound ARS.     

The effect of oligonucleotides complementary to the 3' end of the 16S rRNA on the formation of the bacterial 30S initiation complex

Although much attention was focused on the role of the 16S RNA in mRNA selection by the 30S ribosomal subunit no true consensus site has emerged as yet. Oligonucleotides such as GAGG, UGAU and CCAA which are complementary to the 3' end of the 16S RNA stimulate the AUG-dependent binding of fMet-tRNA to 30S subunits. If those tetranucleotides are used in combination or if the octanucleotides GAGGUGAU and UGAUCCAA are applied, the degree of stimulation remains unchanged. Effects are strictly dependent on the presence of initiation factor 2 (IF-2) and cannot be produced by using A4 or U4. With sequences covalently linked to the AUG as in CCAAAUG and UGAUCCAAAUG, the efficiency of the initiation complex formation decreases significantly as compared to AUG with UGAUCCAAAUG being the least efficient mRNA analogue. The pentadecanucleotide GAGGUGAUCCAAAUG, however, shows the highest efficiency in directing the binding of the fMet-tRNA to 30S subunits and is clearly superior to AUG. Initiation factor 2 (IF-2), which stimulates tRNA binding significantly with AUG and CCAAAUG, both in terms of slope and plateau values of the binding curves, does not effect the initial rate of tRNA binding to GAGGUGAUCCAAAUG. In another set of experiments, where GAGG and AUG are separated by oligo(U) or oligo(A) sequences, the effect of chain length was investigated. mRNA analogues with a spacer of 6-9 nucleotides show the highest binding efficiencies, with a U spacer being superior to an A spacer, indicating that a more flexible spacer favours tRNA binding.     

Inhibitor of translational initiation in sea urchin eggs prevents mRNA utilization

Actively reinitiating cell-free translation systems from sea urchin eggs and embryos have been developed. The extracts retain the overall differences in protein synthetic activity observed in intact eggs and embryos. The effect of combining extracts from eggs and embryos suggests the presence of a dominant inhibitor of translation in the egg. This inhibitor also prevents the initiation of translation in a cell-free system from rabbit reticulocytes. In the reticulocyte system, the egg inhibitor causes the accumulation of 48 S preinitiation complexes, as measured by the accumulation of initiator tRNA and globin mRNA on the small ribosomal subunit. Accumulation of this uncommon intermediate suggests either that the inhibitor prevents the binding of the 60 S ribosomal subunit, or that it prevents migration of the 40 S subunit from the 5' end of mRNA to the first AUG.     

A human autoantibody specific for a unique conserved region of 28 S ribosomal RNA inhibits the interaction of elongation factors 1 alpha and 2 with ribosomes

An autoantibody reactive with a conserved sequence of 28 S rRNA (anti-28 S) was identified in serum from a patient with systemic lupus erythematosus. Anti-28 S protected a unique 59-nucleotide fragment synthesized in vitro against RNase T1 digestion. RNA sequence analysis revealed that it corresponded to residues 1944-2002 in human 28 S rRNA and 1767-1825 in mouse 28 S rRNA. These sequences are identical and highly conserved throughout all known eukaryotic 28 S rRNAs. In addition, this fragment is homologous to residues 1052-1110 of Escherichia coli 23 S rRNA that lies within the GTP hydrolysis center of the 50 S ribosomal subunit. Anti-28 S and its Fab fragments strongly inhibited poly(U)-directed polyphenylalanine synthesis, but had no effect on ribosomal peptidyltransferase activity. This effect resulted from inhibition of the binding of elongation factors EF-1 alpha and EF-2 to ribosomes and of the associated GTP hydrolysis. The inhibitory effect was almost completely suppressed by preincubation of anti-28 S with 28 S rRNA or in vitro synthesized RNA fragments containing the immunoreactive region. These results show that the immunoreactive conserved region of 28 S rRNA participates in the interaction of ribosomes with the two elongation factors in protein synthesis.     

Binding of wheat germ ribosomes to bisulfite-modified reovirus messenger RNA: Evidence for a scanning mechanism

A scanning mechanism has been proposed (Kozak, 1978) to explain how eukaryotic ribosomes select the correct AUG codon for initiation of protein synthesis. The hypothesis is that a 40 S ribosomal subunit binds initially at or near the 5′-terminus of a message and subsequently migrates toward the interior of the messenger RNA, stopping when it encounters the first AUG codon, at which point a 60 S subunit joins and peptide bond formation begins. The scanning mechanism predicts that if a message were modified by introduction of a new AUG triplet upstream of the existing initiator codon, the adventitious AUG should be the preferred site for formation of an 80 S initiation complex. This prediction has been confirmed in the present studies with two reovirus messenger RNAs, in which sodium bisulfite was used to convert an ACG sequence (located in the 5′ untranslated region of each message) to AUG. Analysis of the ribosome-protected mRNA fragments recovered from sparsomycin-blocked 80 S initiation complexes revealed that a high percentage of wheat germ ribosomes were centered around the “unnatural” 5′-proximal AUG created by the bisulfite treatment, although some ribosomes were also positioned at the second (normal) initiator codon. The bisulfite modification was carried out in 7 m-urea at 37 °C. resulting in quantitative conversion of cytosine to uracil. Thus, both the primary and secondary structure of the message were drastically altered. These perturbations did not impair the efficiency of ribosome binding, nor did the highly unfolded state of the mRNA permit ribosomes to attach to spurious sites in the interior of the message. The data support a mechanism in which the initiator codon is selected by virtue of its position in a message (i.e. closest to the 5′-terminus), without regard to either the primary or secondary structure of the flanking regions.     

A snapshot of the 30S ribosomal subunit capturing mRNA via the Shine-Dalgarno interaction

In the initiation phase of bacterial translation, the 30S ribosomal subunit captures mRNA in preparation for binding with initiator tRNA. The purine-rich Shine-Dalgarno (SD) sequence, in the 5' untranslated region of the mRNA, anchors the 30S subunit near the start codon, via base pairing with an anti-SD (aSD) sequence at the 3' terminus of 16S rRNA. Here, we present the 3.3 A crystal structure of the Thermus thermophilus 30S subunit bound with an mRNA mimic. The duplex formed by the SD and aSD sequences is snugly docked in a "chamber" between the head and platform domains, demonstrating how the 30S subunit captures and stabilizes the otherwise labile SD helix. This location of the SD helix is suitable for the placement of the start codon AUG in the immediate vicinity of the mRNA channel, in agreement with reported crosslinks between the second position of the start codon and G1530 of 16S rRNA.     

Initiation of translation with Pseudomonas aeruginosa phage PP7 RNA: nucleotide sequence of the coat cistron ribosome binding site.

Initiation complex formation between PP7 RNA and ribosomes of Pseudomonas aeruginosa and Escherichia coli has been investigated. The PP7 RNA fragments protected by both species of ribosome have been isolated, and their sequences have been determined. Only one binding sites is available on the intact PP7 RNA strand, and this site is recognized by ribosomes of both species. The PP7 RNA binding site is approximately 38 nucleotides long. It contains two AUG sequences and a purine-rich segment near the 5'-end that is complementary to segments near the 3'-ends of the 16S ribosomal RNA's of both P. aeruginosa and E. coli. In order to establish which of the AUG codons acts as the initiator, the H2N-terminal amino acid sequence of PP7 coat protein was determined. This sequence is compatible with the codon sequence following the second AUG codon. The extent of the reaction of PP7 RNA with E. coli ribosomes is greater than with P. aeruginosa ribosomes, but our results do not indicate a qualitative difference in the initial interaction between intact PP7 RNA and the ribosomes of either species.     

Ribosomes bind leaderless mRNA in Escherichia coli through recognition of their 5'-terminal AUG

Leaderless mRNAs are translated in the absence of upstream signals that normally contribute to ribosome binding and translation efficiency. In order to identify ribosomal components that interact with leaderless mRNA, a fragment of leaderless cI mRNA from bacteriophage λ, with a 4-thiouridine (4^S-U) substituted at the +2 position of the AUG start codon, was used to form cross-links to Escherichia coli ribosomes during binary (mRNA+ribosome) and ternary (mRNA+ribosome+initiator tRNA) complex formation. Ribosome binding assays (i.e., toeprints) demonstrated tRNA-dependent binding of leaderless mRNA to ribosomes; however, cross-links between the start codon and 30S subunit rRNA and r-proteins formed independent of initiator tRNA. Toeprints revealed that a leaderless mRNA's 5′-AUG is required for stable binding. Furthermore, the addition of a 5′-terminal AUG triplet to a random RNA fragment can make it both competent and competitive for ribosome binding, suggesting that a leaderless mRNA's start codon is a major feature for ribosome interaction. Cross-linking assays indicate that a subset of 30S subunit r-proteins, located at either end of the mRNA tunnel, contribute to tRNA-independent contacts and/or interactions with a leaderless mRNA's start codon. The interaction of leaderless mRNA with ribosomes may reveal features of mRNA binding and AUG recognition that are distinct from known signals but are important for translation initiation of all mRNAs.