Transfer-messenger RNA


Posted by mmcmanus
presentation: 
evolution and conservation: 

the tmRNA/SmpB system

tmRNA and SmpB can be found in all sequenced eubacterial genomes, as well as in rare plastidial and phage genomes. Fragments of the tmRNA gene have also been found in some mitochondrial genomes, though these fragments lack an ORF encoding a tag sequence. To date, the tmRNA system has not been identified in any nuclear genomes and does not appear to be used by archaea. (8)

tag sequences (the tmRNA open reading frame)

There are now 632 tag sequences posted by Indiana University on the tmRNA wesbite (See external link 1.) Of these sequences, the coding peptide is usually about 10 amino acids long with the C-terminal consensus motif YALAA. In addition, the tag sequences contain a high proportion of alanine and asparagines residues. It has been suggested that amino acid sequences that can be efficiently and accurately translated are overrepresented in tag sequences. Perhaps for this reason, tag sequences tend to exclude easily misread codons or rare and unstable amino acids. (8)

conservation of structure

The tRNA-like domain of tmRNA contains a D-loop, a T-arm and an acceptor stem. As with all tRNA, the 3’ end of the acceptor stem of this domain terminates in CCA. In order to be recognized by alanyl-tRNA synthetase, the acceptor stem also contains a G:U wobble base pair. However, the anticodon stem is absent, with a “connecting” structure linking the tRNA-like domain to the rest of the tmRNA in its place. The rest of the tmRNA is comprised of the ORF of the mRNA-like domain, and two to four pseudoknots. Although all known tmRNAs contain pseudoknots, reason for their conservation remains a mystery, as tmRNA mutants where pseudoknots are replaced by simple hairpin structures do not show a significant loss in tmRNA function.  (1, 8, 9)

conservation of function

Though all eubacteria have been found to possess the tmRNA/SmpB system, its appears to play a role of varying importance throughout the kingdom. In some organisms, such as Neisseria gonorrhoeae, if tmRNA is disrupted, the mutants are unable to survive. Yet if the same mutation is present in E. coli, the population only sees a mild decrease in rate of growth. It is likely that organisms that are less severely affected by loss of function in tmRNA/SmpB have developed an alternative though less efficient mechanism for releasing stalled ribosomes. (8)

 

significant partner RNAs or proteins: 

As mentioned previously, alanyl-tRNA synthetase is required for tmRNA activation, EF-Tu is necessary for ribosome binding, RNase R is needed for degradation of nonstop RNAs, and SmpB is crucial for almost all tmRNA processes.

Degradation of proteins tagged by tmRNAs is accomplished three energy-dependent proteases, ClpXP, ClpAP, FtsH as well as Tsp, a periplasmic energy-independent protease. The majority of degradation is performed by ClpXP in vitro, while FtsH degrades only a small subset of proteins with low thermodynamic stability. Various adaptor proteins are also involved in mediating protein degradation (1, 8)

 

mechanism of action: 

In the current model, the process of trans-translation occurs as follows: First, the alanine-charged tmRNA system recognizes a stalled ribosome. Next, the tmRNA manifests its tRNA-like properties in binding the A-site of the ribosome and donating its alanine to the polypeptide chain. 3) The mRNA-like properties of tmRNA allow it to code for proteolysis tag on the aberrant protein, as well as a stop codon for release of the ribosome. 4) The defective mRNA is degraded. Recognizing and binding a stalled ribosome For a tRNA, EF-Tu and GTP mediate delivery to the ribosome in an active process where GTP is hydrolyzed to form GDP. GDP is then released along with EF-Tu after the correct codon-anticodon interactions to stabilize binding. (1)

For tmRNA a similar process presumably occurs, although the tRNA-like region lacks an anticodon. Instead, SmpB, a roughly 160 residue protein which binds specifically to tmRNA, is necessary for association with EF-Tu, GTP and the A- site of the ribosome. The actual stoichiometric proportions of SmpB, ribosomal subunit and tmRNA binding are still unknown. (1)

It has recently been proposed that the tmRNA complex recognizes stalled ribosomes via steric restrictions. In ribosomes undergoing active translation, both the P and A sites are filled and the 3’ mRNA is associated tightly with ribosomes and RNA polymerase, preventing the bulky tmRNA complex from binding. However, the 3’ ends of nonstop mRNAs remain caught at the P site, leaving the A site open to the tmRNA complex. (8)

tRNA-like functions

When the tmRNA complex is bound to the ribosomal A-site, transpeptidation occurs as in normal peptide synthesis, where the nascent polypeptide is transferred to the tmRNA alanine and shifted to the ribosomal P-site. (1)

mRNA-like functions

Amazingly, translation is then switched from the nonstop mRNA to the tmRNA ORF without a classic AUG start codon, Shine-Dalgarno sequence, or ribosomal reassembly. Though the mechanism by which this occurs is still unknown, through mutagenesis studies, it has been shown that a GCA resume codon and a conserved UAG sequence two nucleotides upstream are necessary for establishing the correct peptide reading frame. (1, 8)

Upon translation of the tmRNA ORF terminating in a proper stop codon, a tag is added to the nascent polypeptide, directing it for targeted proteolysis. The mRNA is then released, and SmpB and tmRNA mediate its degradation by RNase R. (1)

 

cellular functions: 

The tmRNA system has two main functions: 1) rescue of stalled ribosomes and 2) mRNA/protein quality control. This is accomplished in a process called trans-translation.

In normal peptide synthesis, mRNA is translated by ribosomes in a 5’ to 3’ direction until a stop codon is reached. The stop codon serves as a binding site for recruiting a release factor to the A site which cleaves the tRNA-polypeptide bond. The ribosome then disassembles into its 30S and 50S subunits and is recycled back into the pool for more translation. However, in aberrant mRNA sequences lacking stop codons, the ribosome remains stalled and cannot be released. (1,8)

The tmRNA system is able to recognize, bind and release stalled ribosomes, that would otherwise be unusable and decrease the overall efficiency of translation. It also enables degradation of the defective mRNA so that it cannot re-engage active ribosomes. Lastly, it adds a tag to the aberrant protein product, targeting it for proteolysis. (1,8)

functions in bacteria

How commonly is the tmRNA system used in bacteria? In E. coli, tmRNA rescue of ribosomes occurs in 1 out of every 250 translational events. Even in in this species, where the tmRNA system is inessential, tmRNA deletion mutants constitutively activate heat shock response as in a state of stress. For many other bacteria, the tmRNA system is crucial for viability (see Evolution and Conservation: conservation of function). The tmRNA system is also necessary for virulence in pathogenic species such as Salmonella enterica and Yersinia pseudotuberculosis where it increases survival in macrophages. There is also evidence that tmRNA is involved in motility, cell cycle, type III secretion systems and flagellar synthesis. (1)

Recently, tmRNA has also been shown to help maintain low levels of Lac repressor in E. coli. When the Lac repressor tetramer binds its own operator, transcription of the gene is interrupted, forming a nonstop mRNA. In the absence of the tmRNA system, as in the deletion mutants, the resulting Lac repressor fragments are still able to bind operators and repressor activity is increased, only to be amplified with further translation. However, when intact, the tmRNA system degrades the aberrant mRNA and Lac repressor fragments, limiting repressor levels for efficient Lac induction when lactose metabolism may be required later on. (1)

functions in phages

Phages may use tmRNA activity to gauge the translational abilities of the host. For example, when ribosomes in E. coli stall during translation of the hybrid λ-P22 phage Mu lysogen repressor, tmRNA-mediated release mediates the induction of the lysogenic cycle. (1)

 

biogenesis: 

As with pre-tRNAs, pre-tmRNAs are processed by cellular ribonucleases which remove 5’ and 3’ nucleotides. This results in the formation of a mature tmRNA where 5’ and 3’ ends fold together, and the 3’ hydroxyl group at the terminal CCA of the acceptor arm can then be charged with alanine. In some bacteria, e.g. Bacillus subtilis, the 3’ CCA region is added on by tRNA nucleotidyltransferase. In bacterial lineages that have undergone a circular permutation in the tmRNA sequence, an excision event must also occur to form a mature two-piece tmRNA. (8, 9)

Rates of tmRNA biogenesis and degradation are highly influenced by intracellular signals. In E. coli, each cell usually has about 700 tmRNA molecules, and about one per 10-20 ribosomes. However, when its 1:1 binding partner SmpB reaches sub-stoichiometric levels, tmRNA is degraded more quickly. Levels of tmRNA can be dependent on normal cellular processes such as cell cycle, as in Caulobacter cresecentus. Concentrations can also change drastically in response to cellular stress, as in. subtilis where tmRNA can reach 10-fold its normal levels in conditions of heat shock. (8)

 

example members: 

Two types of tmRNAs are known: single-chain tmRNAs and two-piece tmRNAs.  In most eubacteria, a single tmRNA molecule is encoded by the ssrA  gene.  However, in alpha-proteobacteria, beta-proteobacteria, and cyanobacteria, the ssrA gene has undergone a circular permutation, leaving the mature 3' end of the RNA upstream of the mature 5' end.  As a result, in these bacterial lineages tmRNA is actually formed from two distinct RNA molecules. (1)

 

nomenclature: 

Transfer-messenger RNA (tmRNA), also known as 10Sa RNA, is encoded by the gene ssrA.  tmRNA is a molecule of RNA that has dual functions as both a transfer RNA and a messenger RNA. As a tRNA, it recognizes and binds ribosomes stalled by aberrant mRNAs with the help of its protein partner SmpB . As an mRNA, it adds a degradation tag to protein fragments, targeting them for proteolysis. (1)

 

potential as a tool: 

Specific bacteria can be identified in a mixture by creating a fluorescent probe to tmRNA, which is present at high copy numbers in the cell. In the past, probes have been made against 16S rRNA, but it has been difficult to distinguish between closely related bacteria because rRNA sequences is highly conserved across species. In 2001, Schönhuber et al. found that by designing probes after sequence alignment analysis, individual species such as Lactococcus lactus, or general classes such as gram-positive bacteria could be pinpointed using FISH (fluorescent in situ hybridization). (10)

 

discovery: 

a small, stable RNA

In 1979, while analyzing nucleic acid contents of E. coli via polyacrylamide electrophoresis, Ray and Apirion found an RNA about 10S in size and thus named it 10S RNA. However, at this time they found little interaction of 10S RNA with ribosomes and concluded that 10S RNA had no mRNA activity. (2)

In 1982, Jain, et al. actually discovered two distinct RNA molecules in the 10S position of the polyacrylamide gel: 10Sa (now tmRNA) and 10Sb (now M1RNA) (3).

tRNA-like properties

In 1992 Tyagi and Kinger noted that the 3' end of 10Sa RNA isolated from Mycobacterium tuberculosis showed striking sequence alignment with the pseudouridine region of tRNAs of various organisms (4).  In 1994, Komine, et al. discovered that 10Sa RNA could form a structure equivalent to the acceptor stem, D-loop and T-loop of an alanine tRNA (See Figure 1). In vitro, they showed that purified 10Sa RNA could be recognized and charged by alanine. In vivo, they found that defects in cell growth and motility in a 10Sa RNA deletion strain could be rescued by introduction of a plasmid encoding alanyl-tRNA synthetase. (5)

mRNA-like properties

In 1995, Tu, et al. identified mRNA-like properties of 10Sa RNA. While producing and purifying recombinant IL-6 in E. coli, they noticed a fraction of the IL-6 population was C-terminally truncated and followed by identical peptide tags of AANDQDYALAA-COOH. This sequence matched the putative polypeptide encoded by 10Sa RNA, thus the group set out to identify whether 10Sa RNA was responsible for generating the tag. When they disrupted the gene encoding 10Sa RNA in vivo, they found that although the IL-6 population still contained C-terminally truncated members, the characteristic peptide tag was absent. (6)

the dual functions of tmRNA

In 1996, Keiler et al. described the ability of 10Sa RNA to both 1) free ribosomes from messenger RNA sequences lacking stop codons and 2) add a tag to the aberrant peptide, tagging it for degadation. (7)  In 1999, Karzai et al. pinpointed SmpB as a key binding partner to tmRNA in Salmonella enterica mutants that phenocopied tmRNA deletion strains of E. coli (8).

 

Figure 1.  (Public domain from Wikipedia:tmRNA).

tmRNA structure

 

role in human disease: 

The tmRNA system is vital for growth of many pathogenic bacterial species including Mycobacterium pneumoniae, of the same genus as the causative agent of tuberculosis, and Neisseria gonorrheae, the causative agent of the sexually transmitted infection, gonorrhea. Many more bacteria that do not require the tmRNA system to grow under optimal conditions in vitro will exhibit defects without it when under environmental stress. For example, Salmonella typhimurium, an enteric pathogen loses viability in macrophages in the absence of a functional tmRNA system. Yersinia pseudotuberculosis, a relative of the causative agent of plague, is unable to use its type III secretion system within a host without the tmRNA system. Thus unable to deliver effector molecules to target cells it is rendered avirulent, and does not cause disease in mouse models. (1)