TmRNA
Encyclopedia
Transfer-messenger RNA is a bacterial RNA
molecule with dual tRNA-like and messenger RNA
-like properties. The tmRNA forms a ribonucleoprotein
complex (tmRNP) together with Small Protein B (SmpB), Elongation Factor Tu (EF-Tu
), and ribosomal protein
S1. In trans-translation, tmRNA and its associated proteins bind to bacterial ribosomes which have stalled in the middle of protein biosynthesis
, for example when reaching the end of a messenger RNA
which has lost its stop codon. The tmRNA is remarkably versatile: it recycles the stalled ribosome, adds a proteolysis
-inducing tag to the unfinished polypeptide, and facilitates the degradation of the aberrant messenger RNA
. In the majority of bacteria these functions are carried out by standard one-piece tmRNAs. In other bacterial species, a permuted ssrA gene produces a two-piece tmRNA in which two separate RNA chains are joined by base-pairing.
RNA was further resolved into tmRNA and the similarly-sized RNase P
RNA (10Sb). The presence of pseudouridine
in the mixed 10S RNA hinted that tmRNA has modified bases found also in tRNA. The similarity at the 3' end of tmRNA to the T stem-loop of tRNA was first recognized upon sequencing ssrA from Mycobacterium tuberculosis
. Subsequent sequence comparison revealed the full tRNA-like domain (TLD) formed by the 5' and 3' ends of tmRNA, including the acceptor stem with elements like those in alanine tRNA that promote its aminoacylation by alanine-tRNA ligase
. It also revealed differences from tRNA: the anticodon arm is missing in tmRNA, and the D arm
region is a loop without base pairs.
was elucidated by comparative sequence analysis and structural probing
. Watson-Crick and G-U base pairs were identified by comparing the bacterial tmRNA sequences using automated computational methods in combination with manual alignment
procedures. The accompanying figure shows the base pairing pattern of this prototypical tmRNA, which is organized into 12 phylogenetically supported helices (also called pairings P1 to P12), some divided into helical segments.
A prominent feature of every tmRNA is the conserved tRNA-like domain (TLD), composed of helices 1, 12, and 2a (analogs of the tRNA acceptor stem, T-stem and variable stem, respectively), and containing the 5' monophosphate and alanylatable 3' CCA ends. The mRNA-like region (MLR) is in standard tmRNA a large loop containing pseudoknots and a coding sequence (CDS) for the tag peptide
, marked by the resume codon and the stop codon
. The encoded tag peptide (ANDENYALAA in E. coli) varies among bacteria, perhaps depending on the set of proteases and adaptors available.
tmRNAs typically contain four pseudoknot
s, one (pk1) upstream of the tag peptide CDS, and the other three pseudoknots (pk2 to pk4) downstream of the CDS. The pseudoknot regions, although generally conserved, are evolutionarily plasic. For example, in the (one-piece) tmRNAs of cyanobacteria, pk4 is substituted with two tandemly arranged smaller pseudoknots. This suggests that tmRNA folding outside the TLD can be important, yet the pseudoknot region lacks conserved residues and pseudoknots are among the first structures to be lost as ssrA sequences diverge in plastid and endosymbiont lineages. Base pairing in the three-pseudoknot region of E. coli tmRNA is disrupted during trans-translation.
s compared to the four (or more) of standard tmRNA.
Alphaproteobacteria
have two signature sequences: replacement of the typical T-loop sequence TΨCRANY with GGCRGUA, and the sequence AACAGAA in the large loop of the 3´-terminal pseudoknot. In mitochondria, the MLR has been lost, and a remarkable re-permutation of mitochondrial ssrA results in a small one-piece product in Jakoba libera.
The cyanobacteria provide the most plausible case for evolution of a permuted gene from a standard gene, due to remarkable sequence similarities between the two gene types as they occur in different Synechococcus strains.
are most effective. Depending on the bacterial species, the 3'-CCA is either encoded or added by tRNA nucleotidyltransferase
.
Similar processing at internal sites of permuted precursor tmRNA explains its physical splitting into two pieces. The two-piece tmRNAs have two additional ends whose processing must be considered. For alphaproteobacteria, one 5´ end is the unprocessed start site of transcription. The far 3´ end may in some cases be the result of rho-independent termination.
]
High-resolution structures of the complete tmRNA molecules are currently unavailable and may be difficult to obtain due the inherent flexibility of the MLR. In 2007,
the crystal structure of the Thermus thermophilus
TLD bound to the SmpB protein was obtained at 3 Å resolution. This structure shows that SmpB mimics the D stem and the anticodon of a canonical tRNA whereas helical section 2a of tmRNA corresponds to the variable arm of tRNA.
A cryo-electron microscopy
study of tmRNA at an early stage of trans-translation shows the spatial relationship between the ribosome
and the tmRNP (tmRNA bound to the EF-Tu
protein). The TLD is located near the GTPase-associated center in the 50S ribosomal subunit; helix 5 and pseudoknots pk2 to pk4 form an arc around the beak of the 30S ribosomal subunit.
-derived peptides each tagged at the carboxyl termini with the same 11-amino acid residue extension (A)ANDENYALAA. With the exception of the N-terminal alanine
, which comes from the 3' end of tmRNA itself, this tag sequence was traced to a short open reading frame in E. coli tmRNA. Recognizing that the tag peptide confers proteolysis
, the trans-translation model for tmRNA action was proposed.
While details of the trans-translation mechanism are under investigation it is generally agreed that tmRNA first occupies the empty A site of the stalled ribosome
. Subsequently, the ribosome moves from the 3' end of the truncated messenger RNA
onto the resume codon of the MLR, followed by a slippage-prone stage from where translation continues normally until the in-frame tmRNA stop codon
is encountered. Trans-translation is essential in some bacterial species, whereas other bacteria require tmRNA to survive when subjected to stressful growth conditions. Depending on the organism, the tag peptide may be recognized by a variety of proteases or protease adapters.
Non-chromosomal ssrA was first detected in a genomic survey of mycobacteriophages (in 10% of the phages). Other mobile elements
including plasmids and genomic islands have been found bearing ssrA. One interesting case is Rhodobacter sphaeroides ATCC 17025, whose native tmRNA gene is disrupted by a genomic island; unlike all other genomic islands in tmRNA (or tRNA) genes this island has inactivated the native target gene without restoration, yet compensates by carrying its own tmRNA gene. A very unusual relative of ssrA is found in the lytic mycobacteriophage DS6A, that encodes little more than the TLD.
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
molecule with dual tRNA-like and messenger RNA
Messenger RNA
Messenger RNA is a molecule of RNA encoding a chemical "blueprint" for a protein product. mRNA is transcribed from a DNA template, and carries coding information to the sites of protein synthesis: the ribosomes. Here, the nucleic acid polymer is translated into a polymer of amino acids: a protein...
-like properties. The tmRNA forms a ribonucleoprotein
Ribonucleoprotein
Ribonucleoprotein is a nucleoprotein that contains RNA, i.e. it is an association that combines ribonucleic acid and protein together. A few known examples include the ribosome, the enzyme telomerase, vault ribonucleoproteins, and small nuclear RNPs , which are implicated in pre-mRNA splicing and...
complex (tmRNP) together with Small Protein B (SmpB), Elongation Factor Tu (EF-Tu
EF-Tu
EF-Tu is one of the prokaryotic elongation factors.The prokaryotic factor EF-Tu mediates the entry of the aminoacyl-tRNA into a free site of the ribosome. EF-Tu functions by binding an aminoacylated, or charged, tRNA molecule in the cytoplasm...
), and ribosomal protein
Ribosomal protein
A ribosomal protein is any of the proteins that, in conjunction with rRNA, make up the ribosomal subunits involved in the cellular process of translation. A large part of the knowledge about these organic molecules has come from the study of E. coli ribosomes. Most ribosomic proteins have been...
S1. In trans-translation, tmRNA and its associated proteins bind to bacterial ribosomes which have stalled in the middle of protein biosynthesis
Protein biosynthesis
Protein biosynthesis is the process in which cells build or manufacture proteins. The term is sometimes used to refer only to protein translation but more often it refers to a multi-step process, beginning with amino acid synthesis and transcription of nuclear DNA into messenger RNA, which is then...
, for example when reaching the end of a messenger RNA
Messenger RNA
Messenger RNA is a molecule of RNA encoding a chemical "blueprint" for a protein product. mRNA is transcribed from a DNA template, and carries coding information to the sites of protein synthesis: the ribosomes. Here, the nucleic acid polymer is translated into a polymer of amino acids: a protein...
which has lost its stop codon. The tmRNA is remarkably versatile: it recycles the stalled ribosome, adds a proteolysis
Proteolysis
Proteolysis is the directed degradation of proteins by cellular enzymes called proteases or by intramolecular digestion.-Purposes:Proteolysis is used by the cell for several purposes...
-inducing tag to the unfinished polypeptide, and facilitates the degradation of the aberrant messenger RNA
Messenger RNA
Messenger RNA is a molecule of RNA encoding a chemical "blueprint" for a protein product. mRNA is transcribed from a DNA template, and carries coding information to the sites of protein synthesis: the ribosomes. Here, the nucleic acid polymer is translated into a polymer of amino acids: a protein...
. In the majority of bacteria these functions are carried out by standard one-piece tmRNAs. In other bacterial species, a permuted ssrA gene produces a two-piece tmRNA in which two separate RNA chains are joined by base-pairing.
Discovery of tmRNA and early work
tmRNA was first designated 10Sa RNA after a mixed “10S” electrophoretic fraction of Escherichia coliEscherichia coli
Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...
RNA was further resolved into tmRNA and the similarly-sized RNase P
RNase P
Ribonuclease P is a type of ribonuclease which cleaves RNA. RNase P is unique from other RNases in that it is a ribozyme – a ribonucleic acid that acts as a catalyst in the same way that a protein based enzyme would. Its function is to cleave off an extra, or precursor, sequence of RNA on tRNA...
RNA (10Sb). The presence of pseudouridine
Pseudouridine
Pseudouridine is the C-glycoside isomer of the nucleoside uridine, and it is the most prevalent of the over one hundred different modified nucleosides found in RNA. Ψ is found in all species and in many classes of RNA except mRNA...
in the mixed 10S RNA hinted that tmRNA has modified bases found also in tRNA. The similarity at the 3' end of tmRNA to the T stem-loop of tRNA was first recognized upon sequencing ssrA from Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis is a pathogenic bacterial species in the genus Mycobacterium and the causative agent of most cases of tuberculosis . First discovered in 1882 by Robert Koch, M...
. Subsequent sequence comparison revealed the full tRNA-like domain (TLD) formed by the 5' and 3' ends of tmRNA, including the acceptor stem with elements like those in alanine tRNA that promote its aminoacylation by alanine-tRNA ligase
Alanine-tRNA ligase
In enzymology, an alanine-tRNA ligase is an enzyme that catalyzes the chemical reactionThe 3 substrates of this enzyme are ATP, L-alanine, and tRNA, whereas its 3 products are AMP, diphosphate, and L-alanyl-tRNA....
. It also revealed differences from tRNA: the anticodon arm is missing in tmRNA, and the D arm
D arm
The D arm is a feature in the tertiary structure of transfer RNA . It is composed of the two D stems and the D loop. The D loop contains the base dihydrouracil. The D loop's main function is that of recognition...
region is a loop without base pairs.
Secondary structure of the standard one-piece tmRNAs
The complete E. coli tmRNA secondary structureSecondary structure
In biochemistry and structural biology, secondary structure is the general three-dimensional form of local segments of biopolymers such as proteins and nucleic acids...
was elucidated by comparative sequence analysis and structural probing
Protein structure prediction
Protein structure prediction is the prediction of the three-dimensional structure of a protein from its amino acid sequence — that is, the prediction of its secondary, tertiary, and quaternary structure from its primary structure. Structure prediction is fundamentally different from the inverse...
. Watson-Crick and G-U base pairs were identified by comparing the bacterial tmRNA sequences using automated computational methods in combination with manual alignment
Sequence alignment
In bioinformatics, a sequence alignment is a way of arranging the sequences of DNA, RNA, or protein to identify regions of similarity that may be a consequence of functional, structural, or evolutionary relationships between the sequences. Aligned sequences of nucleotide or amino acid residues are...
procedures. The accompanying figure shows the base pairing pattern of this prototypical tmRNA, which is organized into 12 phylogenetically supported helices (also called pairings P1 to P12), some divided into helical segments.
A prominent feature of every tmRNA is the conserved tRNA-like domain (TLD), composed of helices 1, 12, and 2a (analogs of the tRNA acceptor stem, T-stem and variable stem, respectively), and containing the 5' monophosphate and alanylatable 3' CCA ends. The mRNA-like region (MLR) is in standard tmRNA a large loop containing pseudoknots and a coding sequence (CDS) for the tag peptide
Peptide
Peptides are short polymers of amino acid monomers linked by peptide bonds. They are distinguished from proteins on the basis of size, typically containing less than 50 monomer units. The shortest peptides are dipeptides, consisting of two amino acids joined by a single peptide bond...
, marked by the resume codon and the stop codon
Stop codon
In the genetic code, a stop codon is a nucleotide triplet within messenger RNA that signals a termination of translation. Proteins are based on polypeptides, which are unique sequences of amino acids. Most codons in messenger RNA correspond to the addition of an amino acid to a growing polypeptide...
. The encoded tag peptide (ANDENYALAA in E. coli) varies among bacteria, perhaps depending on the set of proteases and adaptors available.
tmRNAs typically contain four pseudoknot
Pseudoknot
A pseudoknot is a nucleic acid secondary structure containing at least two stem-loop structures in which half of one stem is intercalated between the two halves of another stem. The pseudoknot was first recognized in the turnip yellow mosaic virus in 1982...
s, one (pk1) upstream of the tag peptide CDS, and the other three pseudoknots (pk2 to pk4) downstream of the CDS. The pseudoknot regions, although generally conserved, are evolutionarily plasic. For example, in the (one-piece) tmRNAs of cyanobacteria, pk4 is substituted with two tandemly arranged smaller pseudoknots. This suggests that tmRNA folding outside the TLD can be important, yet the pseudoknot region lacks conserved residues and pseudoknots are among the first structures to be lost as ssrA sequences diverge in plastid and endosymbiont lineages. Base pairing in the three-pseudoknot region of E. coli tmRNA is disrupted during trans-translation.
Two-piece tmRNAs
Circularly permuted ssrA has been reported in three major lineages: i) all alphaproteobacteria and the primitive mitochondria of jakobid protists, ii) two disjoint groups of cyanobacteria (Gloeobacter and a clade containing Prochlorococcus and many Synechococcus), and iii) some members of the betaproteobacteria (Cupriavidus and some Rhodocyclales). All produce the same overall two-piece (acceptor and coding pieces) form, equivalent to the standard form nicked downstream of the reading frame. None retain more than two pseudoknotPseudoknot
A pseudoknot is a nucleic acid secondary structure containing at least two stem-loop structures in which half of one stem is intercalated between the two halves of another stem. The pseudoknot was first recognized in the turnip yellow mosaic virus in 1982...
s compared to the four (or more) of standard tmRNA.
Alphaproteobacteria
Alphaproteobacteria
Alphaproteobacteria is a class of Proteobacteria. Like all Proteobacteria, they are Gram-negative.-Characteristics:The Alphaproteobacteria comprise most phototrophic genera, but also several genera metabolising C1-compounds , symbionts of plants and animals, and a group of pathogens, the...
have two signature sequences: replacement of the typical T-loop sequence TΨCRANY with GGCRGUA, and the sequence AACAGAA in the large loop of the 3´-terminal pseudoknot. In mitochondria, the MLR has been lost, and a remarkable re-permutation of mitochondrial ssrA results in a small one-piece product in Jakoba libera.
The cyanobacteria provide the most plausible case for evolution of a permuted gene from a standard gene, due to remarkable sequence similarities between the two gene types as they occur in different Synechococcus strains.
tmRNA processing
Most tmRNAs are transcribed as larger precursors which are processed much like tRNA. Cleavage at the 5´ end is by ribonuclease P. Multiple exonucleases can participate in the processing of the 3´ end of tmRNA, although RNase T and RNase PHRNase PH
RNase PH is an 3'-5' exoribonuclease and nucleotidyltransferase, present in archaea and bacteria, that is involved in tRNA processing. Contrary to hydrolytic enzymes, it is a phosphorolytic enzyme, meaning that it uses inorganic phosphate as a cofactor to cleave nucleotide-nucleotide bonds,...
are most effective. Depending on the bacterial species, the 3'-CCA is either encoded or added by tRNA nucleotidyltransferase
TRNA nucleotidyltransferase
In enzymology, a tRNA nucleotidyltransferase is an enzyme that catalyzes the chemical reactionThus, the two substrates of this enzyme are tRNA and phosphate, whereas its two products are tRNA and nucleoside diphosphate....
.
Similar processing at internal sites of permuted precursor tmRNA explains its physical splitting into two pieces. The two-piece tmRNAs have two additional ends whose processing must be considered. For alphaproteobacteria, one 5´ end is the unprocessed start site of transcription. The far 3´ end may in some cases be the result of rho-independent termination.
Three-dimensional structures
]]
High-resolution structures of the complete tmRNA molecules are currently unavailable and may be difficult to obtain due the inherent flexibility of the MLR. In 2007,
the crystal structure of the Thermus thermophilus
Thermus thermophilus
Thermus thermophilus is a Gram negative eubacterium used in a range of biotechnological applications, including as a model organism for genetic manipulation, structural genomics, and systems biology. The bacterium is extremely thermophilic, with an optimal growth temperature of about...
TLD bound to the SmpB protein was obtained at 3 Å resolution. This structure shows that SmpB mimics the D stem and the anticodon of a canonical tRNA whereas helical section 2a of tmRNA corresponds to the variable arm of tRNA.
A cryo-electron microscopy
Cryo-electron microscopy
Cryo-electron microscopy , or electron cryomicroscopy, is a form of transmission electron microscopy where the sample is studied at cryogenic temperatures...
study of tmRNA at an early stage of trans-translation shows the spatial relationship between the ribosome
Ribosome
A ribosome is a component of cells that assembles the twenty specific amino acid molecules to form the particular protein molecule determined by the nucleotide sequence of an RNA molecule....
and the tmRNP (tmRNA bound to the EF-Tu
EF-Tu
EF-Tu is one of the prokaryotic elongation factors.The prokaryotic factor EF-Tu mediates the entry of the aminoacyl-tRNA into a free site of the ribosome. EF-Tu functions by binding an aminoacylated, or charged, tRNA molecule in the cytoplasm...
protein). The TLD is located near the GTPase-associated center in the 50S ribosomal subunit; helix 5 and pseudoknots pk2 to pk4 form an arc around the beak of the 30S ribosomal subunit.
Trans-translation
Coding by tmRNA was discovered in 1995 when Simpson and coworkers overexpressed a mouse cytokine in E. coli and found several truncated cytokineCytokine
Cytokines are small cell-signaling protein molecules that are secreted by the glial cells of the nervous system and by numerous cells of the immune system and are a category of signaling molecules used extensively in intercellular communication...
-derived peptides each tagged at the carboxyl termini with the same 11-amino acid residue extension (A)ANDENYALAA. With the exception of the N-terminal alanine
Alanine
Alanine is an α-amino acid with the chemical formula CH3CHCOOH. The L-isomer is one of the 20 amino acids encoded by the genetic code. Its codons are GCU, GCC, GCA, and GCG. It is classified as a nonpolar amino acid...
, which comes from the 3' end of tmRNA itself, this tag sequence was traced to a short open reading frame in E. coli tmRNA. Recognizing that the tag peptide confers proteolysis
Proteolysis
Proteolysis is the directed degradation of proteins by cellular enzymes called proteases or by intramolecular digestion.-Purposes:Proteolysis is used by the cell for several purposes...
, the trans-translation model for tmRNA action was proposed.
While details of the trans-translation mechanism are under investigation it is generally agreed that tmRNA first occupies the empty A site of the stalled ribosome
Ribosome
A ribosome is a component of cells that assembles the twenty specific amino acid molecules to form the particular protein molecule determined by the nucleotide sequence of an RNA molecule....
. Subsequently, the ribosome moves from the 3' end of the truncated messenger RNA
Messenger RNA
Messenger RNA is a molecule of RNA encoding a chemical "blueprint" for a protein product. mRNA is transcribed from a DNA template, and carries coding information to the sites of protein synthesis: the ribosomes. Here, the nucleic acid polymer is translated into a polymer of amino acids: a protein...
onto the resume codon of the MLR, followed by a slippage-prone stage from where translation continues normally until the in-frame tmRNA stop codon
Stop codon
In the genetic code, a stop codon is a nucleotide triplet within messenger RNA that signals a termination of translation. Proteins are based on polypeptides, which are unique sequences of amino acids. Most codons in messenger RNA correspond to the addition of an amino acid to a growing polypeptide...
is encountered. Trans-translation is essential in some bacterial species, whereas other bacteria require tmRNA to survive when subjected to stressful growth conditions. Depending on the organism, the tag peptide may be recognized by a variety of proteases or protease adapters.
Mobile genetic elements and the tmRNA gene
ssrA is both a target for some mobile DNAs and a passenger on others. It has been found interrupted by three types of mobile elements. By different strategies none of these disrupt gene function: group I introns remove themselves by self-splicing, rickettsial palindromic elements (RPEs) insert in innocuous sites, and integrase-encoding genomic islands split their target ssrA yet restore the split-off portion.Non-chromosomal ssrA was first detected in a genomic survey of mycobacteriophages (in 10% of the phages). Other mobile elements
Transposon
Transposable elements are sequences of DNA that can move or transpose themselves to new positions within the genome of a single cell. The mechanism of transposition can be either "copy and paste" or "cut and paste". Transposition can create phenotypically significant mutations and alter the cell's...
including plasmids and genomic islands have been found bearing ssrA. One interesting case is Rhodobacter sphaeroides ATCC 17025, whose native tmRNA gene is disrupted by a genomic island; unlike all other genomic islands in tmRNA (or tRNA) genes this island has inactivated the native target gene without restoration, yet compensates by carrying its own tmRNA gene. A very unusual relative of ssrA is found in the lytic mycobacteriophage DS6A, that encodes little more than the TLD.