Sandbox 1: Difference between revisions

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==History==

Hfq was first identified in the lates 1960s in Escherichia coli as an essential host factor for the RNA replication of the bacteriophage Qβ. Later, in the 1990s, was shown that Hfq could provide greatest benefits to the bacterium itself, rather than to its phage predator. Tsui and colleagues, in 1994, described a diverse of pleiotropic effects caused by the disruption of the hfq gene, related to fitness reduce, stress response impairment and, in pathogenic bacterias, diminishment of virulence.

Hfq was first identified in the lates 1960s in Escherichia coli as an essential host factor for the RNA replication of the bacteriophage Qβ. Later, in the 1990s, was shown that Hfq could provide greatest benefits to the bacterium itself, rather than to its phage predator. Tsui and colleagues, in 1994, described a diverse of pleiotropic effects caused by the disruption of the hfq gene, related to fitness reduce, stress response impairment and, in pathogenic bacterias, diminishment of virulence (Vogel & Luisi, 2011).

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Hfq is a bacterial post-transcriptional regulator that modulates RNA structure, acting as a RNA chaperone. The most well-characterized function of Hfq is its role as a RNA matchmaker, promoting interactions between trans-encoded sRNAs (small regulatory RNAs) and their messenger RNAs (mRNAs) targets, leading translation and RNA stability regulation (Santos et al., 2019). In Bacteria sRNAs are critical for bacterial survival under adverse conditions and expression of virulence factors. Trans-encoded sRNAs are a large class of this regulators that are transcribed from a different locus than their targets and act as a imperfect base pairing, often regulating multiple mRNA (Faner & Feig, 2014).

The resulting network of regulation of Hfq-RNA are best characterized in Escherichia coli and Salmonella typhimurium, but it importance is recognized in a diverse population of bacteria (Faner & Feig, 2014).

The resulting network of regulation of Hfq-RNA are best characterized in ''Escherichia coli'' and ''Salmonella typhimurium'', but it importance is recognized in a diverse population of bacteria (Faner & Feig, 2014).

Basically, Hfq plays a active role in positioning RNAs for optimal base pairing by changing the secondary or tertiary structures of RNAs, bringing RNAs into proximity, neutralizing the negative charge of the two pairing RNAs, stimulating the nucleation of the first base pairs as well as facilitating the further annealing of the two RNA strands (Updegrove et al., 2016).

There are several mechanisms to Hfq-mediated regulation. Most of the Hfq/RNAs/RNAm interactions described report inhibition of translation, although there are cases of positive regulation (Santos et al., 2019). Hfq can suppress protein synthesis by aiding the sRNA to bind the 5’ region of its target mRNA turning this region inaccessible to translation initiation. In the other way, this RNA chaperone can also act boosting translation, where sRNA guided by Hfq can bind in the 5’ region of the mRNA, changing its structure that otherwise inhibits ribosome binding. Hfq can also present a sRNA to its mRNA target leading to both degradation (Vogel & Luisi, 2011). It also has been shown that Hfq can interacts with either RNAs or RNAm independently, not just between this molecules. Hfq can modulate mRNA stability by directly binding and remodeling without the sRNA. It can also promotes the polyadenylation at the 3’ end of mRNAs, which in turn triggers 3’ to 5’ degradation by a exoribonuclease. The Hfq association also protects sRNAs from degradative activity of polynucleotide phosphorylase (PNPase) and ribonuclease E (RNase E) (Vogel & Luisi, 2011; Santos et al., 2019).

Hfq is able to bind ATP, however, the chaperoning activity does not require ATP hydrolysis (Santos et al., 2019).

[[Image:~~Hfq_functions~~.PNG]]

[[Image:Hfq funções.PNG]]

[[Image:Hfq funções2.PNG]]

Recently, the discovery of others binding substrates with Hfq has expanded the regulatory spectrum of this protein. It was found that this RNA chaperone can also bind with rRNA and tRNA. Studies have shown that Hfq promotes ribosome assembly in bacteria, where this protein is required for the processing of pre-16S and folding of the mature 16S rRNA in E. coli, displaying Hfq as a important ribosome biogenesis factor in bacterias. This process seems to be independent of Hfq interactions with sRNAs, which the binding surfaces of the vast majority of the Hfq-dependent sRNAs are the proximal and rim faces, and for the rRNA, in the distal face of the Hfq. The same was observed for tRNA, where Hfq was also associated as a important pre-tRNA maturation factor, however, the binding of the structural elements of tRNAs occurs through the proximal face of Hfq (Santos et al., 2019).

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[[Image:~~Funções~~.~~png~~]]

[[Image:Hfq functions(2).PNG]]

==Structure Highlights==

The structure of S. aureus protein has a size of 8.9 kDa. Hfq forms a symmetric hexameric ring with a diameter of ~ 65 Å and width of 23 Å. This hexamer has a central hole, doughnut shape like. The protein has a N-terminal alfa helix (α1) in each subunit tracked by five B strands (β1-β5) Hfq contains a sm fold who share conserved amino acids like the aspartic acid 40 and the glycine 34, determining hydrophobic residues present in the Sm1 motif which maintains the highly distorted Sm1 fold. Tyr56 and Tyr63, highly conserved in the Sm2 motif, are fundamental for the interaction between subunits. The glutamine 8 and tyrosine 42 are highly conserved in Hfq proteins due to their role in uracil binding.

The structure of S. aureus protein has a size of 8.9 kDa. Hfq forms a symmetric hexameric ring with a diameter of ~ 65 Å and width of 23 Å. This hexamer has a central hole, doughnut shape like. The protein has a N-terminal alfa helix (α1) in each subunit tracked by five antiparallel B strands (β1-β5) Hfq contains a sm fold who share conserved amino acids like the aspartic acid 40 and the glycine 34, determining hydrophobic residues present in the Sm1 motif which maintains the highly distorted Sm1 fold. Tyr56 and Tyr63, highly conserved in the Sm2 motif, are fundamental for the interaction between subunits. The glutamine 8 and tyrosine 42 are highly conserved in Hfq proteins due to their role in uracil binding.

Since Sm proteins often yield inactive hexameric forms when over-expressed in bacteria (Zaric et al. 2005) and the heptameric form has never been observed in Hfq, it is likely that the hexamer is the thermodynamically more stable form of this fold.

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[[Image:Estrutura.png]]

[[Image:Hfq_structure(legend).PNG]]

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[[Image:Hfq-RNA_binding.PNG]]

The crystal structure of ''Staphylococcus aureus'' Hfq-RNA complex (1KQ2) was important to reveal how the proximal RNA binding site can bind to an oligonucleotide with internal uridines (5’-AUUUUUG-3’). In this structure, the six-nucleotide binding pockets of the Hfq ring are occupied by the first six residues of the oligonucleotide, whereas the 3′-terminal guanine is expelled (Weichenrieder, 2014).

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@@ Line 4: / Line 4: @@
 ==History==
-Hfq was first identified in the lates 1960s in Escherichia coli as an essential host factor for the RNA replication of the bacteriophage Qβ. Later, in the 1990s, was shown that Hfq could provide greatest benefits to the bacterium itself, rather than to its phage predator. Tsui and colleagues, in 1994, described a diverse of pleiotropic effects caused by the disruption of the hfq gene, related to fitness reduce, stress response impairment and, in pathogenic bacterias, diminishment of virulence.
+Hfq was first identified in the lates 1960s in Escherichia coli as an essential host factor for the RNA replication of the bacteriophage Qβ. Later, in the 1990s, was shown that Hfq could provide greatest benefits to the bacterium itself, rather than to its phage predator. Tsui and colleagues, in 1994, described a diverse of pleiotropic effects caused by the disruption of the hfq gene, related to fitness reduce, stress response impairment and, in pathogenic bacterias, diminishment of virulence (Vogel & Luisi, 2011).
@@ Line 11: / Line 11: @@
 Hfq is a bacterial post-transcriptional regulator that modulates RNA structure, acting as a RNA chaperone. The most well-characterized function of Hfq is its role as a RNA matchmaker, promoting interactions between trans-encoded sRNAs (small regulatory RNAs) and their messenger RNAs (mRNAs) targets, leading translation and RNA stability regulation (Santos et al., 2019). In Bacteria sRNAs are critical for bacterial survival under adverse conditions and expression of virulence factors. Trans-encoded sRNAs are a large class of this regulators that are transcribed from a different locus than their targets and act as a imperfect base pairing, often regulating multiple mRNA (Faner & Feig, 2014).
-The resulting network of regulation of Hfq-RNA are best characterized in Escherichia coli and Salmonella typhimurium, but it importance is recognized in a diverse population of bacteria (Faner & Feig, 2014).
+The resulting network of regulation of Hfq-RNA are best characterized in ''Escherichia coli'' and ''Salmonella typhimurium'', but it importance is recognized in a diverse population of bacteria (Faner & Feig, 2014).
 Basically, Hfq plays a active role in positioning RNAs for optimal base pairing by changing the secondary or tertiary structures of RNAs, bringing RNAs into proximity, neutralizing the negative charge of the two pairing RNAs, stimulating the nucleation of the first base pairs as well as facilitating the further annealing of the two RNA strands (Updegrove et al., 2016).
 There are several mechanisms to Hfq-mediated regulation. Most of the Hfq/RNAs/RNAm interactions described report inhibition of translation, although there are cases of positive regulation (Santos et al., 2019). Hfq can suppress protein synthesis by aiding the sRNA to bind the 5’ region of its target mRNA turning this region inaccessible to translation initiation. In the other way, this RNA chaperone can also act boosting translation, where sRNA guided by Hfq can bind in the 5’ region of the mRNA, changing its structure that otherwise inhibits ribosome binding. Hfq can also present a sRNA to its mRNA target leading to both degradation (Vogel & Luisi, 2011). It also has been shown that Hfq can interacts with either RNAs or RNAm independently, not just between this molecules. Hfq can modulate mRNA stability by directly binding and remodeling without the sRNA. It can also promotes the polyadenylation at the 3’ end of mRNAs, which in turn triggers 3’ to 5’ degradation by a exoribonuclease. The Hfq association also protects sRNAs from degradative activity of polynucleotide phosphorylase (PNPase) and ribonuclease E (RNase E) (Vogel & Luisi, 2011; Santos et al., 2019).
 Hfq is able to bind ATP, however, the chaperoning activity does not require ATP hydrolysis (Santos et al., 2019).
-[[Image:Hfq_functions.PNG]]
+[[Image:Hfq funções.PNG]]
+[[Image:Hfq funções2.PNG]]
 Recently, the discovery of others binding substrates with Hfq has expanded the regulatory spectrum of this protein. It was found that this RNA chaperone can also bind with rRNA and tRNA. Studies have shown that Hfq promotes ribosome assembly in bacteria, where this protein is required for the processing of pre-16S and folding of the mature 16S rRNA in E. coli, displaying Hfq as a important ribosome biogenesis factor in bacterias. This process seems to be independent of Hfq interactions with sRNAs, which the binding surfaces of the vast majority of the Hfq-dependent sRNAs are the proximal and rim faces, and for the rRNA, in the distal face of the Hfq. The same was observed for tRNA, where Hfq was also associated as a important pre-tRNA maturation factor, however, the binding of the structural elements of tRNAs occurs through the proximal face of Hfq (Santos et al., 2019).
@@ Line 22: / Line 23: @@
-[[Image:Funções.png]]
+[[Image:Hfq functions(2).PNG]]
 ==Structure Highlights==
-The structure of S. aureus protein has a size of 8.9 kDa. Hfq forms a symmetric hexameric ring with a diameter of ~ 65 Å and width of 23 Å. This hexamer has a central hole, doughnut shape like. The protein has a N-terminal alfa helix (α1) in each subunit tracked by five B strands (β1-β5) Hfq contains a sm fold who share conserved amino acids like the aspartic acid 40 and the glycine 34,  determining hydrophobic residues present in the Sm1 motif which maintains the highly distorted Sm1 fold. Tyr56 and Tyr63, highly conserved in the Sm2 motif, are fundamental for the interaction between subunits. The glutamine 8 and tyrosine 42 are highly conserved in Hfq proteins due to their role in uracil binding.
+The structure of S. aureus protein has a size of 8.9 kDa. Hfq forms a symmetric hexameric ring with a diameter of ~ 65 Å and width of 23 Å. This hexamer has a central hole, doughnut shape like. The protein has a N-terminal alfa helix (α1) in each subunit tracked by five antiparallel B strands (β1-β5) Hfq contains a sm fold who share conserved amino acids like the aspartic acid 40 and the glycine 34,  determining hydrophobic residues present in the Sm1 motif which maintains the highly distorted Sm1 fold. Tyr56 and Tyr63, highly conserved in the Sm2 motif, are fundamental for the interaction between subunits. The glutamine 8 and tyrosine 42 are highly conserved in Hfq proteins due to their role in uracil binding.
 Since Sm proteins often yield inactive hexameric forms when over-expressed in bacteria (Zaric et al. 2005) and the heptameric form has never been observed in Hfq, it is likely that the hexamer is the thermodynamically more stable form of this fold.
@@ Line 38: / Line 39: @@
 [[Image:Estrutura.png]]
+[[Image:Hfq_structure(legend).PNG]]
@@ Line 51: / Line 53: @@
 [[Image:Hfq-RNA_binding.PNG]]
+The crystal structure of ''Staphylococcus aureus'' Hfq-RNA complex (1KQ2) was important to reveal how the proximal RNA binding site can bind to an oligonucleotide with internal uridines (5’-AUUUUUG-3’). In this structure, the six-nucleotide binding pockets of the Hfq ring are occupied by the first six residues of the oligonucleotide, whereas the 3′-terminal guanine is expelled (Weichenrieder, 2014).