What is the Role of Trna During Translation Apex

What is the Role of Trna During Translation Apex.

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Annu Rev Microbiol.
Author manuscript; bachelor in PMC 2015 Dec 17.

Published in final edited form as:

PMCID:

PMC4682898

NIHMSID:

NIHMS743581

Regulation of Translation Initiation by RNA Binding Proteins

Paul Babitzke

1Department of Biochemistry and Molecular Biological science, The Pennsylvania Land University, University Park, Pennsylvania 16802

Ballad South. Baker

1Department of Biochemistry and Molecular Biology, The Pennsylvania State University, Academy Park, Pennsylvania 16802

Tony Romeo

iiDepartment of Microbiology and Prison cell Science, University of Florida, Gainesville, Florida 32611

Abstract

RNA binding proteins are capable of regulating translation initiation past a diversity of mechanisms. Although the vast majority of these regulatory mechanisms involve translational repression, 1 example of translational activation has been characterized in item. The RNA recognition targets of these regulatory proteins exhibit a wide range in structural complexity, with some proteins recognizing complex pseudoknot structures and others bounden to simple RNA hairpins and/or curt repeated single-stranded sequences. In some instances the bound poly peptide directly competes with ribosome binding, and in other instances the bound poly peptide promotes formation of an RNA construction that inhibits ribosome bounden. Examples also exist in which the spring protein traps the ribosome in a circuitous that is incapable of initiating translation.

Keywords:

repression, activation, autoregulation, protein-RNA interaction, RNA structure

INTRODUCTION

Bacteria and their viruses are capable of rapid alterations in cistron expression in response to changing ecology and physiological conditions. Factor expression in leaner is regulated at many levels, including transcription, translation, and mRNA stability. Translation initiation in bacteria begins with the germination of a binary complex consisting of the 30S ribosomal subunit and either mRNA or fmet-tRNAf
Met. The binary complex is converted to a preternary circuitous following binding of the other RNA molecule; withal, the two RNA molecules in the preternary complex exercise not interact with 1 some other. Conversion of the preternary complex to the translation-competent ternary complex involves proper interaction of mRNA with fmet-tRNAf
Met
(32). The Smoothen-Dalgarno (SD) sequence in mRNA base-pairs with the anti-SD sequence in 16S rRNA within the 30S subunit and thereby correctly positions the initiation codon in the ribosome. Efficient translation often leads to increased mRNA stability, as translating ribosomes tin protect the mRNA from nucleolytic attack.

Studies on the regulation of protein synthesis have shown that RNA structural features present in the 5′-untranslated leaders tin influence translation initiation in bacteria (32, 47, 77). A diverseness of translational command mechanisms that involve regulated access of ribosomes to mRNA in bacteria and bacteriophages have been identified. It is well documented that RNA secondary structures can sequester the SD sequence and forestall ribosome bounden. Protein-contained mechanisms in which the kinetics of RNA folding plays a crucial office in regulating the rate of SD-sequestering hairpin formation have been characterized (64). Poly peptide-dependent germination (xiv), stabilization (33, 42), or destabilization (27, 72) of SD sequestering hairpins controls translation initiation of a variety of genes. Several examples exist in which an RNA binding protein tin direct compete with ribosome binding. Most of these cases involve a structured RNA recognition target (eight, 10, 33, 42, 45), although there are examples in which a sequence-specific RNA binding protein can compete with ribosome bounden (six, 19).

This review describes some of the best-characterized examples in which RNA bounden proteins specifically control translation initiation of their cognate mRNA targets. In virtually cases, the protein either direct or indirectly competes with 30S ribosomal subunit binding to the mRNA; still, two examples have been identified in which protein-mRNA interaction entraps the 30S subunit in an inactive complex (17, 48). Most all known examples of poly peptide-dependent translational control are repression mechanisms; however, one bacterial translation activation machinery has been described in item (26).

TRANSLATIONAL REPRESSION: FEEDBACK REGULATION

Translation of nearly
Escherichia coli
ribosomal protein genes is regulated past autoregulatory feedback mechanisms in which one gene in the operon encodes a ribosomal protein (r-protein) that tin bind either to rRNA during ribosome biogenesis or to its mRNA and repress translation (31, 77). The alternative r-poly peptide binding sites are similar in structure and hence constitute interesting examples of molecular mimicry. During rapid growth the r-poly peptide binds to its target in newly synthesized rRNA; withal, nether unfavorable growth weather condition rRNA synthesis is reduced such that free r-protein instead binds to its mRNA target. The bound r-protein typically inhibits translation of one cistron in the mRNA, while the downstream cistrons are often repressed via translational coupling, a procedure in which translation of a downstream factor is at to the lowest degree partially dependent on translation of the cistron only upstream. In several cases repression occurs past a competition mechanism in which the r-protein prevents 30S ribosomal subunit bounden. In contrast, two entrapment mechanisms in which the regulatory r-protein functions by trapping the 30S subunit in an inactive complex with the mRNA have been identified (31, 77). Feedback regulation is not restricted to r-protein genes. An
E. coli
tRNA synthetase enzyme that can either charge its cognate tRNAs or bind to its own mRNA and repress translation has been identified (44).

Eastward. coli
L20

The L35 operon of
Due east. coli
(rpmI-rplT) encodes r-proteins L35 and L20 (31, 77). The translational repressor protein L20, encoded by
rplT, binds to its leader transcript and competes with 30S ribosomal subunit binding (25). Spring L20 directly represses L35 synthesis, while translation of its ain gene is inhibited via translational coupling. The regulatory region of this operon is particularly long (~450 nt) and includes several stem-loop structures, as well as a long-range pseudoknot. This pseudo-knot forms between the loop of one hairpin and a single-stranded segment simply upstream from the
rpmI
SD sequence (x). L20 is capable of bounden to one of two sites in the leader transcript: the pseudoknot structure (binding site 1) and a bulged stem-loop (binding site two) positioned a few bases upstream from the
rpmI
SD sequence. Both binding sites are similar to the L20 bounden site in 23S rRNA (Effigy i
a

) (23). Although both binding sites appear to be important for autogenous control, L20 is unable to bind to both sites simultaneously (1). Because the stem-loop of bounden site 2 is capable of forming prior to the pseudoknot containing binding site 1, a translation control model in which L20 first binds to binding site 2 has been proposed. Once the pseudoknot structure forms, a repressing complex forms betwixt L20 and the mRNA, although it is not known which of the ii binding sites would exist occupied past L20 (25). A construction of the repressing circuitous would probable answer this question.


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Feedback regulation of
E. coli
ribosomal poly peptide operons. Structures of the mRNA (a–e) and rRNA (a–d
) bounden sites for each protein are shown. (a) L20 binding sites in the
rpmI
leader and 23S rRNA transcripts (23). (b) S8 binding sites in the
spc
and 16S rRNA transcripts (39). (c) L10-(L12)iv
binding sites in the
rplJ
leader and 23S rRNA transcripts (29). (d
) S15 binding sites in the
rpsO
and 16S rRNA transcripts (17). (due east) Pseudoknot involved in S4 binding to the
rpsM
transcript (48).

E. coli
S8

The
spc
operon of
E. coli
(rplNXE-rpsNH-rplFR-rpsE-rpmD-rplO-secY-rpmJ) encodes 11 r-proteins and SecY, a component of the secretion apparatus (31, 77). The translation re-pressor protein S8, encoded past
rpsH, binds to the
rplE
translation initiation region and represses L5 synthesis by competing with 30S ribosomal subunit binding. Repression is extended to several of the downstream cistrons via translational coupling. The S8 bounden site in
rplE
is included within a bulged RNA hairpin that begins just upstream of the initiation codon (8). Although the structure of this hairpin is similar to that of the S8 binding site in 16S rRNA (Figure 1
b

), the analogousness of S8 for its rRNA target is about fivefold higher (21). Two extra bulged nucleotides within the mRNA target (+2 and +5,
Figure 1
b

) reduce the affinity of S8 for its mRNA target (71). Crystal structures of
Methanococcus jannaschii
S8 jump to a 16S rRNA fragment (59), as well as
E. coli
S8 jump to a synthetic mRNA target, have been reported (39). The two structures are highly like despite the sequence differences of the two RNAs. S8 binds to 1 face of the constructed mRNA target, the internal loop and the last loop; even so, the
spc
operon mRNA does non include a concluding loop equivalent to the synthetic mRNA target (39). Thus, it appears that the most relevant interaction from the crystal structure is the packing of an antiparallel β-canvass in the C-terminal domain of S8 against the minor grove of the internal loop, equally side chains inside this β-sheet make the only two base-specific hydrogen bail contacts in the entire complex. The two extra bulged nucleotides that reduce affinity of S8 for
spc
mRNA had no result on the conformation of the RNA that interacts with S8 (39). Although the structural basis for reduced affinity of S8 for it smRNA target remains unresolved, the S8-mRNA structure is consistent with a model in which spring S8 would compete with 30S subunit bounden.

E. coli
L10-(L12)four

The L10 operon of
East. coli
(rplJL) encodes r-proteins L10 and L12 (31, 77). The translation repressor of the L10 operon consists of a complex of one L10 subunit and 4 L12 subunits. The L10-(L12)four
complex binds to the
rplJL
leader ~150 bases upstream from the
rplJ
initiation codon and directly represses translation of the showtime cistron. However, the mechanism past which the spring complex represses translation at such a considerable altitude has not been identified. A recent modeling and RNA mutagenesis study identified fundamental features of the binding targets in 23S rRNA and mRNA (29). The minimal mRNA recognition structure, which is conserved amidst several bacterial species, includes a kink-turn motif and a UUAA burl loop sequence (Figure 1
c

) (xi, 29). Both of these elements are also role of the 23S rRNA bounden target, although the structural context of the UUAA sequence differs; the recognized loop in rRNA is a U-turn motif (Effigy 1
c

). Nevertheless, the dissimilar structural contexts of the loops obviously evolved so that they contribute similarly to the L10-(L12)iv
binding analogousness (29). While the repressor complex binds to both RNAs with comparable affinity, cooperative interaction of L11 and the L10-(L12)four
complex with the rRNA target leads to increased affinity of the L10-(L12)iv
circuitous for 23S rRNA, thereby providing an caption for how ribosomes affectively compete with mRNA for L10-(L12)4
binding (29). The location of putative L10-(L12)iv
mRNA binding sites in a variety of bacterial species suggests that similar autoregulatory mechanisms might control expression of their respective operons; however, L10-(L12)4-mediated regulation has but been reported for
Due east. coli
(29).

E. coli
S4

The α operon of
East. coli
(rpsMKD-rpoD-rplQ) encodes four ribosomal subunits and the α-subunit of RNA polymerase. Ribosomal subunit S4, encoded by
rpsD, binds to the mRNA leader and directly inhibits translation of
rpsM
(31, 77). The binding site for S4 consists of a nested pseudoknot structure that includes the SD sequence and translation initiation region; the pseudoknot extends from virtually −75 to +35 nt relative to the
rpsM
initiation codon (Figure 1
east

) (48). Results from RNA toeprint and electrophoretic mobility experiments indicated that the leader RNA is capable of undergoing a conformational switch between active and inactive states of the pseudoknot (48, 56). Although 30S ribosomal subunits are capable of binding to both conformations, only the agile conformation is expert for translation (57). S4 binds only the inactive mRNA conformation and traps 30S ribosomal subunits in a dead-end complex that is unable to bind initiator tRNA (56). This repression machinery is unusual in that S4 functions every bit an allosteric effector that drives the equilibrium between two conformation states of the RNA toward the inactive land. Moreover, S4 acts at a 2nd irreversible step leading to entrapment of the 30S subunit in an inactive complex with mRNA (48). As structural information of S4 interaction with its mRNA target is not bachelor, the critical contacts of this r-poly peptide with the pseudoknot structure have not been firmly established. Withal, the finding that both S4 and 30S subunits tin simultaneously demark to the mRNA provides strong prove that the S4 binding site does non include the
rpsD
SD sequence and initiation codon. A similar translation repression machinery may be responsible for decision-making expression of the α operon of
Bacillus subtilis, although details of the S4-mediated translation repression machinery were non investigated in this organism (22).

E. coli
S15

The S15 operon of
East. coli (rpsO-pnp)
encodes r-poly peptide S15 and PNPase, a 3′ to 5′ exoribonuclease (13, 31, 77). The translation repressor protein S15 binds to a region that contains the
rpsO
translation initiation region. Every bit in the case of S4-mediated translational repression, S15 binds to a pseudoknot structure in the mRNA and blocks translation via an entrapment mechanism. Crystal structures of the
Thermus thermophilus
30S ribosomal subunits (49, 68) allowed modeling of
East. coli
S15 with 16S rRNA (52). S15 recognizes two sites in 16S rRNA that are close to i some other (Figure ane
d

). Site 1 is subdivided into subsites 1a and 1b. Subsite 1a is part of a three-way helical junction, and subsite 1b is formed by one of the stems. Site 2 consists of adjacent GU and GC base pairs and is located near subsite 1b on the same stem (17). The pseudoknot of
rpsO
mRNA consists of two stacked helices that are bridged by a single adenine, and the SD sequence and AUG initiation codon are contained within a large connecting loop that is not function of the S15 binding site (Figure i
d

). The S15 binding sites in 16S rRNA and
rpsO
mRNA share the GU and GC base pairs of site 2 but boosted sequence and/or structural conservation is non readily apparent. Footprinting and mutagenesis studies of both S15 and its binding site in
rpsO
mRNA identified important contacts involved in S15 binding (17, 53). The GC base pair of the GU/GC motif in both mRNA and rRNA is recognized by His41 and Asp48 of S15. Although the details of how S15 recognizes the GU pair are not firmly established, recognition of the GU/GC motif in mRNA and rRNA is like (17). A likely interaction unique to
rpsO
mRNA is between Arg57 and the single A residue that bridges the ii stacked helices of the pseudo-knot. As mentioned above, binding of S15 to
rpsO
mRNA does not forestall 30S subunit binding but instead traps the 30S subunit in a nonproductive circuitous. A recent cryo-electron microscopy study identified the S15-rpsO
mRNA docking site on the ribosome. In one case S15 dissociates from the complex, the mRNA unfolds such that productive outset codon–initiator tRNA interactions can have identify (36).

E. coli
ThrRS

Threonyl-tRNA synthetase (ThrRS), the enzyme responsible for charging tRNAThr, represses translation of its own gene (thrS) by competing with ribosome binding (44). The
thrS
translation initiation and autoregulatory regions consist of four domains (Figure two). Domain 1 contains the initiation codon and the SD sequence, domains 2 and four consist of RNA hairpins, and domain three is a unmarried-stranded segment separating the two hairpins.
thrS
is unusual in that it contains a dissever ribosome bounden site consisting of domain ane and part of domain 3 (45). ThrRS functions as a homodimer that can demark to two tRNAThr
molecules for aminoacylation. Alternatively, ThrRS can demark to the domain 2 and 4 hairpins inside its own leader transcript. Binding of these ii culling substrates provides an exquisite case of molecular mimicry, as both of the
thrS
leader hairpins closely resemble the an-ticodon loop of the tRNA substrate (Figure two). The starting time base of the anticodon-like loops in
thrS
mRNA can exist inverse without agin affects on regulation, whereas changing the second or tertiary position eliminates repression (43). This blueprint of mRNA recognition mirrors that of the tRNAThr
isoacceptors for which the first position of the anticodon can exist C, K, or U, whereas the second and third positions must be Thousand and U, respectively (44). Crystal structures of ThrRS complexed with two tRNAThr
molecules (46) or two domain 2 binding sites (lx) indicate that recognition of the anticodon and anticodon-similar loops involves interaction with the same amino acids within the C-terminal domain of the protein. In addition, the courage of the tRNA anticodon stem and the stem of domain 2 bind in a similar fashion to the catalytic domain of ThrRS. Biochemical and mutagenesis studies betoken that domain 4 is recognized in the same way as domain 2 (43, 44). Although the binding sites for ThrRS and 30S ribosomal subunits do not strictly overlap, bound ThrRS competes with ribosome binding (44). A cocrystal construction of the
T. thermophilus
ribosome spring to a fragment of
thrS
mRNA provides an explanation of how ThrRS could block ribosome bounden. Molecular modeling indicates that a steric disharmonism betwixt the North-terminal domain of ThrRS and the 30S subunit would forestall simultaneous binding of ThrRS and the ribosome to the same
thrS
mRNA molecule (xxx). In support of this model, deletion of the N-terminal domain of ThrRS prevents translational regulation in vivo. Furthermore, the truncated form of the enzyme was unable to compete with ribosome binding, despite its ability to demark to its mRNA target in vitro (7).


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Feedback regulation of
Escherichia coli thrS. In the absence of bound ThrRS, ribosomes can demark to the carve up ribosome binding site (domains 1 and 3) and initiate translation of
thrS. Dimeric ThrRS can bind to two tRNAThr
molecules for aminoacylation. Alternatively, ThrRS can bind to 2 sites in
thrS
(domains 2 and 4), which inhibits ribosome binding and represses translation. The ThrRS binding sites in
thrS, as well as
thrS
Shine-Dalgarno (SD) sequence and initiation codon, are shown. The anticodon loop of tRNAThr
is shown for comparing (44). The critical anticodon (tRNAThr) and anticodon-similar (thrS) residues are shown.

Every bit is the case with r-proteins of
Due east. coli, the synthesis of most aminoacyl-tRNA synthetases is dependent on growth rate (44). If growth charge per unit increases, the synthesis of tRNAThr
increases, and ThrRS binds to its tRNA substrate rather than to its mRNA. As a consequence, synthesis of ThrRS increases. Yet, when growth charge per unit slows the synthesis of tRNAThr
decreases such that free ThrRS can bind to its own mRNA and repress translation.

TRANSLATIONAL REPRESSION: STRUCTURED AUTOREGULATORY BINDING SITES

Autogenous translational repression mechanisms in which the RNA binding target includes the proteins ain SD sequence have been identified. These bounden sites are often chosen translational operators. The binding site in these systems typically contains an RNA structure with critical unmarried-stranded residues. In some instances additional protein contacts are made with downstream unmarried-stranded sequences. In many means these mechanisms are reminiscent of feedback regulation, with the important distinction that the proteins do not have an alternative rRNA or tRNA binding site.

RNA Phage Coat Proteins

Qβ, MS2, and PP7 are RNA phages whose glaze proteins serve equally structural proteins of the phage capsid. Alternatively, these proteins bind to replicase mRNA and repress translation by competing with ribosome binding, thereby switching from viral replication to virion associates (33, 42, 69). Each binding site forms a hairpin that sequesters the cognate SD sequence and/or initiation codon. Although the RNA structures are similar, the specificity of recognition by the cognate proteins involves the length of the stem, the size and sequence of the loop, and the loop’s position on the stem (Figure 3a
). Furthermore, the importance of the bulged A residue varies; information technology is essential in MS2 but not in Qβ (28, 69). The glaze proteins bind to RNA as dimers and the amino acid residues important for binding are located on a big β-sheet. The crystal structure of the MS2 protein bound to its RNA target indicates that the protein contacts seven phosphate groups on the 5′ side of the hairpin, A and U residues of the loop, and the bulged A balance (63). SELEX experiments with the PP7 coat protein identified a preferred bounden site containing a 4-bp lower stalk, the bulged A residue, a 4-bp upper stem, and a conserved six-base loop at the apex of the construction (33). The PP7 coat protein-RNA cocrystal structure indicates that the starting time three purines in the loop form a purine stack that continues the base stacking of the helical stem (ix). The A residues in the bulge and initiation codon are recognized by nearly identical interactions fabricated past symmetric pockets on the protein dimer. This organisation contrasts with the MS2 coat poly peptide-RNA structure in which both of these pockets are independent within the aforementioned subunit of the dimer (nine).


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Autogenous translational repression of mRNAs containing structured binding sites. The bound regulatory protein competes with ribosome binding in each case. Poly peptide binding sites and/or critical residues involved in protein-RNA interaction are highlighted. Shine-Dalgarno (SD) sequences and initiation codons (Met) of the mRNAs are shown. (a) Binding sites for the coat proteins of bacteriophages Qβ, MS2, and PP7 (33). (b) Binding site for bacteriophage T4 gp32 (5). (c) Binding site for bacteriophage T4 gp43 (40). (d) Binding site for
Mycobacterium smegmatis
PyrR. The altitude between the putative SD sequence and the AUG codon (xiii nt) is unusually long (18).

Bacteriophage T4 gp32

The bacteriophage T4 ssDNA binding protein (gp32) is a zinc-finger protein that functions in phage DNA replication, repair, and recombination. This poly peptide also binds to its own mRNA and represses its translation (37, 55). gp32 kickoff binds to an RNA pseudoknot upstream of its SD sequence, followed by cooperative binding of gp32 to the downstream sequence, which includes four UUAAA/UAAA repeats between the pseudoknot and the initiation codon (Figure iii
b

) (5, 37, 55). Both the pseudoknot and the unstructured echo region are essential for autoregulation (37). Although the bounden of gp32 to ssDNA is non sequence specific, it has a college analogousness for ssDNA than for its mRNA target. The crystal structure of gp32 complexed with ssDNA indicates that the phosphate backbone contacts an electropositive cleft of the protein (54). As a structure with RNA is not bachelor, it is not known how the poly peptide interacts with RNA; however, it is unlikely that the same protein cleft could bind specifically to the pseudoknot and the downstream repeats.

Bacteriophage T4 gp43

The T4 DNA polymerase, gp43, functions in phage DNA replication and every bit an RNA binding protein responsible for repressing its ain translation (ii). The gp43 mRNA target includes a hairpin and a single-stranded tail, which includes the SD sequence (Figure 3
c

) (40). Thus, leap protein competes with ribosome binding. Germination of the hairpin and the length of the tail (sequence-independent) contribute to the specificity and the stability of gp43-RNA interaction. Specificity determinants in the hairpin include the A-C dinucleotide in the hairpin loop and the restricted positioning of purine and pyrimidine residues in the stem (40). It is of historical interest to note that SELEX was first adult for gp43-RNA interaction studies; the RNA variants selected had an affinity similar to that of the natural target (61).

1000. smegmatis
PyrR

PyrR of
B. subtilis
regulates expression of the pyrimidine biosynthetic (pyr) operon by a well-characterized transcription attenuation machinery. When activated by uridine nucleotides, PyrR binds to three positions in the polycistronic transcript, including the leader region upstream of
pyrR, between
pyrR
and
pyrP, and between
pyrP
and
pyrB
(62). In each case, bound PyrR stabilizes an anti-antiterminator structure called the binding loop. Thus, bound PyrR promotes transcription termination in three regions of the
pyr
transcript when sufficient levels of uridine nucleotides are present in the prison cell. Regulation of
pyr
gene expression in
M. smegmatis
past exogenous uracil occurs by a translational repression mechanism rather than by transcription attenuation. In this case, the PyrR binding loop apparently functions as an SD-sequestering hairpin (Figure 3
d

) (18). Bound PyrR stabilizes this construction and competes with 30S ribosomal subunit binding (62).

TRANSLATIONAL REPRESSION: Single-STRANDED Bounden SITES

B. subtilis
TRAP

The
B. subtilis trp
RNA binding attenuation protein (TRAP) plays a central role in controlling tryptophan synthesis and transport past sensing the concentration of tryptophan in the cell. TRAP regulates tryptophan metabolism past participating in transcription attenuation and translational repression mechanisms (xix). In the transcription attenuation mechanism of the
trpEDCFBA
biosynthetic operon, tryptophan-activated TRAP binds to the nascent transcript and promotes germination of an intrinsic terminator past blocking formation of an overlapping antiterminator structure. In the absence of TRAP bounden, the antiterminator structure forms and the operon is expressed. Tryptophan-activated TRAP besides represses translation of
trpE, trpG
(tryptophan synthesis),
trpP
(tryptophan transport), and
ycbK
(putative efflux poly peptide). In the example of
trpE, TRAP bounden promotes germination of a
trpE
SD-sequestering hairpin (14). In dissimilarity, TRAP regulates translation of
trpG, trpP, and
ycbK
directly by bounden to RNA segments that overlap their cognate SD sequences and/or translation initiation regions (xv, 74, 75).

TRAP is a sequence-specific unmarried-stranded RNA bounden protein that binds to trinucleotide repeats (GAG > UAG > AAG >CAG), which are separated past ii to three nonconserved spacer nucleotides (Figure 4). The TRAP binding sites in the
trp
leader,
trpG, trpP, and
ycbK
transcripts contain 9–11 repeats. TRAP consists of 11 identical subunits bundled in a band, with tryptophan binding between next subunits via a network of hydrogen bonds and stacking interactions (19). Eleven repeated KKR motifs on the perimeter of TRAP are crucial for the interaction of tryptophan-activated TRAP with RNA (76). The crystal structure of a
B. stearothermophilus
TRAP-RNA complex revealed that the RNA wraps around the exterior of the protein ring (3). The phosphodiester backbone is on the exterior of the RNA ring, with the bases pointing in toward the protein. The KKR motifs grade hydrogen bonds with the NAG repeats; Lys37 hydrogen bonds to the A balance and both Lys56 and Arg58 hydrogen bail with the third Grand of each repeat.


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Translational repression of
Bacillus subtilis
tryptophan metabolism genes. (a) Model of the
trpE
translational repression mechanism. RNA polymerase pausing during transcription provides additional time for binding of tryptophan-activated
trp
RNA bounden attenuation poly peptide (TRAP). In the absence of bound TRAP (limiting tryptophan), the RNA adopts a construction such that the
trpE
Smooth-Dalgarno (SD) sequence is single stranded and available for ribosome bounden. In the presence of excess tryptophan, TRAP binding promotes formation of the
trpE
SD-sequestering hairpin. (b) TRAP binding sites in the
trpG, trpP, and
ycbK
transcripts. In each case, bound TRAP competes with ribosome binding and represses translation. The SD sequence and initiation codon (Met) for each mRNA is marked.

The TRAP binding site in the
trp
leader consists of 11 repeats. In the
trpE
translational repression machinery, TRAP binding to these repeats promotes formation of the
trpE
SD-sequestering hairpin, which inhibits TrpE synthesis by preventing ribosome bounden (Figure 4
a

) (14). Translation of
trpD
is besides controlled by the
trpE
SD-sequestering hairpin via translational coupling (nineteen). Remarkably, the TRAP bounden site ends ~100 nt upstream from the
trpE
SD sequence. In the absence of bound TRAP, an culling RNA structure forms betwixt a portion of the TRAP binding site (anti-anti-SD sequence) and the anti-SD sequence such that the SD sequence is unmarried-stranded and bachelor for ribosome binding. The general transcription elongation factors NusA and NusG stimulate RNA polymerase pausing during transcription of the
trp
leader, thereby providing boosted fourth dimension for TRAP to demark and promote formation of the
trpE
SD-sequestering hairpin (73).

A third TRAP-dependent regulatory mechanism is responsible for controlling translation initiation of
trpG, trpP, and
ycbK
in which bound TRAP direct blocks 30S ribosomal subunit bounden (15, 74, 75). The TRAP binding site in
trpG
includes nine triplet repeats that overlap its SD sequence (Figure 4
b

). The TRAP binding site in the
trpP
transcript also contains ix triplet repeats; however, in this case the TRAP binding site overlaps the
trpP
SD sequence and extends into the
trpP
coding sequence. In the case of
ycbK, all nine triplet repeats are downstream from the SD sequence and extend further into the
ycbK
coding sequence. Thus, the unusual machinery of TRAP-RNA interaction in which multiple triplet repeats extend over a long linear distance allows the poly peptide to specifically bind to a subset of translation initiation regions and repress translation.

TRAP is found in a few other closely related gram-positive organisms. In each instance, a putative TRAP binding site is positioned in the
trp
operon leader, suggesting that TRAP is capable of controlling expression of these operons by attenuation and/or translational repression mechanisms (24). While experimental back up for transcription attenuation exists for some of these organisms, translational repression by an SD-sequestering hairpin has not been demonstrated for any organism other than
B. subtilis
(19).

Bacteriophage T4 RegA

T4 RegA is a unmarried-stranded RNA binding protein that represses translation of several T4 genes, including its own mRNA, by competing with ribosome binding to the cognate translation initiation region. Although the mRNA targets are AU rich, a consensus bounden site has not been identified (66). While RegA exists as a dimer when gratuitous in solution, this poly peptide binds RNA as a monomer (41). RegA’s RNA bounden domain consists of a surface pocket formed by residues on two loops and an N-final α-helix (20). T4 RegA is 78% identical to that from bacteriophage RB69. SELEX studies identified high-affinity RNA targets for both RegA proteins: AAAAUUGUUAUGUAA for T4 and UAA repeat sequence UAAUAAUAAUAAUAAUA for RB69 (6, 12). Both RegA proteins exhibit a bureaucracy of affinities for their cognate RNAs: gene 44 > gene 45 > regA (51). Thus, the relative affinities of RegA for target sequences explain its chapters to regulate translation of a diverseness of genes prior to repressing its own synthesis.

TRANSLATIONAL REPRESSION: MULTIPLE BINDING SITES

E. coli
CsrA

The
Eastward. coli
carbon storage regulator (CsrA) protein is the key component of a global regulatory system that both represses translation of genes that are induced upon the approach to stationary-stage growth and activates genes that are expressed during exponential-stage growth.
csrA
is broadly distributed among eubacteria, and information technology regulates virulence factors, quorum sensing, move, carbon metabolism, peptide uptake, and biofilm development in diverse species (4). CsrA-mediated repression typically involves binding of this protein to multiple sites, one of which overlaps the cognate SD sequence. Thus, spring CsrA competes with 30S ribosomal subunit bounden. In contrast, the mechanism(southward) of CsrA-mediated activation has non been elucidated. CsrA activity is controlled past its interaction with two noncoding sRNAs (CsrB and CsrC) that comprise multiple binding sites that enable them to sequester and antagonize CsrA (35, 67). In this example of molecular mimicry, the CsrA bounden sites of CsrB and CsrC resemble those of mRNA target molecules (Figure 5). The homeostatic Csr circuitry is controlled by two negative feedback loops: (a) CsrA somehow activates signaling past a two-component signal transduction system (BarA-UvrY) that activates transcription of the sRNA antagonists, CsrB/C. (b) CsrA represses expression of the CsrD protein, which forth with RNase E mediates specific turnover of CsrB/C (58).


An external file that holds a picture, illustration, etc.
Object name is nihms743581f5.jpg

CsrA binding sites in CsrC and
pgaA
leader transcripts. (a) CsrC contains thirteen putative CsrA binding sites (numbered
) and functions equally a CsrA antagonist. Considering several of the binding sites have slight overlaps, only the highly conserved GGA motifs are colored to improve clarity. (b) The
pgaA
leader transcript contains six CsrA binding sites (numbered), 3 of which are present in RNA hairpins. Bound CsrA competes with ribosome bounden and represses translation. The
pgaA
Shine-Dalgarno (SD) sequence and initiation codon (Met) are shown. A high-analogousness SELEX-derived RNA target is shown for comparison. Annotation that this motif is AGA in binding site iv.

CsrA binds to unmarried-stranded sequences present in unstructured RNA or in the loops of brusk hairpins. Binding sites in mRNAs, CsrB/C, and SELEX-derived ligands contain an most universal GGA sequence bracketed by boosted conserved residues (Figure 5). Substitution analysis of a SELEX-derived target confirmed that the GGA motif is critical for high-affinity binding and that secondary structure of the stem-loop was beneficial just non essential for binding (16). Structural studies of three CsrA orthologs have shown that this protein forms a homodimer composed of 5 interdigitated β-strands of each polypeptide and protruding C-terminal α-helices. Critical amino acids that contact bound RNA are located on the β-1 and β-5 strands of opposing polypeptides, which lie together and in parallel on each side of the poly peptide, and on the base of operations of the α-helix. Thus, an identical RNA bounden surface is found on each side of this symmetrical protein dimer. The disquisitional GG residues of RNA come in contact with R44 and V42, the amino acid residues most important for RNA bounden (38, 50).

CsrA target transcripts incorporate betwixt 1 (hfq) and ~20 (CsrB) bounden sites. Typically, at least 2 CsrA binding sites are nowadays in the mRNA leader of a repressed gene, one of which invariably overlaps the SD sequence. Cooperative binding is often observed for transcripts containing multiple binding sites. CsrA targets resemble SD sequences, thus mutation of the SD sequence to promote CsrA binding is relatively facile and likely facilitated the evolutionary expansion of the Csr regulon. Six CsrA binding sites are present in the
pgaA
mRNA leader, two of which overlap the SD sequence and initiation codon (Figure 5
b

) (65). For
pgaA
and other mRNA leaders containing a CsrA bounden hairpin shut to the SD sequence, it is likely that ane RNA binding surface of CsrA first binds with loftier affinity to the stem-loop and the 2d surface bridges to the SD sequence, with the germination of a repression loop. Translational repression by CsrA is normally associated with decreased mRNA stability. The mechanism for altered mRNA stability may be passively related to the inhibition of translation and/or directly associated with nucleolytic cleavage and turnover due to the presence of spring CsrA. Finally, the CsrA-CsrB ribonucle-oprotein complex is globular in form and contains ~1 CsrA dimer per two binding sites in CsrB (35), suggesting that CsrA dimers might tether nonadjacent binding sites in the complex.

TRANSLATIONAL ACTIVATION

Bacteriophage Mu Com

Although there are numerous examples of translational repression mechanisms, examples of translational activation are rare. Investigation of the
com-mom
operon of bacteriophage Mu has provided one instance of an RNA binding poly peptide-mediated translational activation machinery.
mom
encodes a Dna modification enzyme that protects Mu Deoxyribonucleic acid from a diversity of restriction enzymes (26). Translation of
mom
is activated by Com (control of
mom), a zinc-finger RNA binding protein. In the absence of Com, the intercistronic region of the
com-mom
transcript adopts a repressive secondary construction that sequesters the
mom
initiation codon and a portion of the
mom
SD sequence (Effigy half dozen). Com bounden destabilizes the inhibitory structure such that Mom synthesis can proceed (27, 72). Results from mutagenesis studies indicate that the Com bounden site includes both secondary and primary structural features (70). Although the precise structure of the RNA target bound past Com has not been firmly established, Com can demark to a 19-nt sequence containing the hairpin, as shown in
Effigy half-dozen
(34).


An external file that holds a picture, illustration, etc.
Object name is nihms743581f6.jpg

Translational activation model of the bacteriophage Mu
mom
transcript. In the absence of bound Com poly peptide, the RNA adopts an RNA hairpin that sequesters the
mom
Shine-Dalgarno (SD) sequence and initiation codon (Met). Spring Com stabilizes an alternative RNA hairpin such that the
mom
SD sequence and initiation codon are single-stranded and available for ribosome binding (26).

SUMMARY POINTS

  1. RNA binding proteins can repress translation initiation by a wide diverseness of mechanisms, including directly competing with 30S ribosomal subunits, promoting formation of an SD-sequestering hairpin, and entrapping 30S subunits in an inactive complex on mRNA.

  2. Autogenous repression of translation is particularly common in
    E. coli
    r-protein operons and in certain bacteriophages.

  3. Translational activation by RNA binding proteins is rare.

  4. The size of the regulon for an RNA binding protein can range from i to several genes.

  5. The complexity of RNA recognition targets ranges from complex pseudoknot structures to single-stranded triplet repeats, and the number of divide binding sites in target transcripts ranges from 1 to ~20.

ACKNOWLEDGMENTS

This work was supported by grants GM52840 (Lead), GM59969 (TR and Atomic number 82), and GM66794 (TR) from the National Institutes of Health.

Glossary

SD Shine-Dalgarno
Secondary construction intramolecular base-pairing interactions within an RNA molecule
SD-sequestering hairpin an inhibitory RNA secondary construction that contains the SD sequence of a factor
Pseudoknot an RNA secondary structure in which part of ane stem is intercalated betwixt two halves of another stem
SELEX systematic evolution of ligands by exponential enrichment
TRAP trp
RNA bounden attenuation poly peptide
Csr carbon storage regulator
Com control of
mom

Footnotes

DISCLOSURE Argument

The authors are non aware of any affiliations, memberships, funding, or financial holdings that might be perceived equally affecting the objectivity of this review.

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