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Antibiotic resistance is becoming an increasing global health concern. The bacterial protein synthesis machinery, including the ribosome and its associated factors, are the target of over half of the clinically relevant antibiotics currently in use, highlighting the importance of cellular protein production as antibiotic target. The ribosome associated factors HflX and YchF are members of the GTPase superfamily. Their cellular functions are only poorly understood. The overarching goal of this thesis was to determine the location where HflX and YchF bind on the bacterial ribosome. Data presented here confirm that YchF interacts with the ribosomal A-site, while HflX is unique within the GTPase family being able to bind to both the ribosomal A-site and E-site. From this data and subsequent biochemical and biophysical studies, a mechanism for both HflX and YchF function during protein synthesis, specifically under stress conditions, is proposed.

Antibiotic resistance is becoming an increasing global health concern. The bacterial protein synthesis machinery, including the ribosome and its associated factors, are the target of over half of the clinically relevant antibiotics currently in use, highlighting the importance of cellular protein production as antibiotic target. The ribosome associated factors HflX and YchF are members of the GTPase superfamily. Their cellular functions are only poorly understood. The overarching goal of this thesis was to determine the location where HflX and YchF bind on the bacterial ribosome. Data presented here confirm that YchF interacts with the ribosomal A-site, while HflX is unique within the GTPase family being able to bind to both the ribosomal A-site and E-site. From this data and subsequent biochemical and biophysical studies, a mechanism for both HflX and YchF function during protein synthesis, specifically under stress conditions, is proposed.

Antibiotic resistance is becoming an increasing global health concern. The bacterial protein synthesis machinery, including the ribosome and its associated factors, are the target of over half of the clinically relevant antibiotics currently in use, highlighting the importance of cellular protein production as antibiotic target. The ribosome associated factors HflX and YchF are members of the GTPase superfamily. Their cellular functions are only poorly understood. The overarching goal of this thesis was to determine the location where HflX and YchF bind on the bacterial ribosome. Data presented here confirm that YchF interacts with the ribosomal A-site, while HflX is unique within the GTPase family being able to bind to both the ribosomal A-site and E-site. From this data and subsequent biochemical and biophysical studies, a mechanism for both HflX and YchF function during protein synthesis, specifically under stress conditions, is proposed.

The creation of eukaryotic ribosomes is a complex process involving hundreds of assembly factors, including the small nucleolar RNA snR30. Through interacting with the unprocessed precursor ribosomal RNA (pre-rRNA), snR30 is essential for the maturation of pre-rRNA forming the small ribosomal subunit. To characterize snR30’s interaction network, an in vitro approach was used to quantify the affinity of snR30 to the core H/ACA protein complex and the PIN endonuclease Utp23 revealing very tight interactions with affinities in the sub- and low-nanomolar ranges, respectively. Furthermore, the snR30 complex binds tightly to its primary binding site in ribosomal RNA called the C1 site, but only weakly to other predicted interaction sites. Utp23 binds tightly to its predicted interaction site in ribosomal RNA only if adjacent rRNA elements are also present. In conclusion, these findings serve as foundational work to further explore the mechanisms of snR30 during maturation of rRNA.

Pseudouridine synthases are RNA modification enzymes targeting various forms of RNA in the cell. RluB and RluF are pseudouridine synthases modifying adjacent uridines in E. coli ribosomal RNA. These enzymes have a highly conserved RNA-binding loop, distinct between RluB and RluF, proposed to play a role in their specificity. This thesis provides preliminary insight into the importance of the eight-residue binding loops of RluB and RluF for RNA target recognition. I have shown that altering the residues of the binding loop decreases the enzyme’s affinity for RNA and effectively abolish the enzyme’s pseudouridylation activity. I conclude that the binding loops of RluB and RluF are critical for RNA binding and for positioning the target RNA for catalysis. Better understanding the requirements for specificity and target recognition of RluB and RluF will enhance our knowledge on ribosome formation and may aid in engineering new enzymes to pseudouridylate other functional RNAs.

Antibiotic resistance is becoming an increasing global health concern. The bacterial protein synthesis machinery, including the ribosome and its associated factors, are the target of over half of the clinically relevant antibiotics currently in use, highlighting the importance of cellular protein production as antibiotic target. The ribosome associated factors HflX and YchF are members of the GTPase superfamily. Their cellular functions are only poorly understood. The overarching goal of this thesis was to determine the location where HflX and YchF bind on the bacterial ribosome. Data presented here confirm that YchF interacts with the ribosomal A-site, while HflX is unique within the GTPase family being able to bind to both the ribosomal A-site and E-site. From this data and subsequent biochemical and biophysical studies, a mechanism for both HflX and YchF function during protein synthesis, specifically under stress conditions, is proposed.

The creation of eukaryotic ribosomes is a complex process involving hundreds of assembly factors, including the small nucleolar RNA snR30. Through interacting with the unprocessed precursor ribosomal RNA (pre-rRNA), snR30 is essential for the maturation of pre-rRNA forming the small ribosomal subunit. To characterize snR30’s interaction network, an in vitro approach was used to quantify the affinity of snR30 to the core H/ACA protein complex and the PIN endonuclease Utp23 revealing very tight interactions with affinities in the sub- and low-nanomolar ranges, respectively. Furthermore, the snR30 complex binds tightly to its primary binding site in ribosomal RNA called the C1 site, but only weakly to other predicted interaction sites. Utp23 binds tightly to its predicted interaction site in ribosomal RNA only if adjacent rRNA elements are also present. In conclusion, these findings serve as foundational work to further explore the mechanisms of snR30 during maturation of rRNA.

Antibiotic resistance is becoming an increasing global health concern. The bacterial protein synthesis machinery, including the ribosome and its associated factors, are the target of over half of the clinically relevant antibiotics currently in use, highlighting the importance of cellular protein production as antibiotic target. The ribosome associated factors HflX and YchF are members of the GTPase superfamily. Their cellular functions are only poorly understood. The overarching goal of this thesis was to determine the location where HflX and YchF bind on the bacterial ribosome. Data presented here confirm that YchF interacts with the ribosomal A-site, while HflX is unique within the GTPase family being able to bind to both the ribosomal A-site and E-site. From this data and subsequent biochemical and biophysical studies, a mechanism for both HflX and YchF function during protein synthesis, specifically under stress conditions, is proposed.

The creation of eukaryotic ribosomes is a complex process involving hundreds of assembly factors, including the small nucleolar RNA snR30. Through interacting with the unprocessed precursor ribosomal RNA (pre-rRNA), snR30 is essential for the maturation of pre-rRNA forming the small ribosomal subunit. To characterize snR30’s interaction network, an in vitro approach was used to quantify the affinity of snR30 to the core H/ACA protein complex and the PIN endonuclease Utp23 revealing very tight interactions with affinities in the sub- and low-nanomolar ranges, respectively. Furthermore, the snR30 complex binds tightly to its primary binding site in ribosomal RNA called the C1 site, but only weakly to other predicted interaction sites. Utp23 binds tightly to its predicted interaction site in ribosomal RNA only if adjacent rRNA elements are also present. In conclusion, these findings serve as foundational work to further explore the mechanisms of snR30 during maturation of rRNA.

The creation of eukaryotic ribosomes is a complex process involving hundreds of assembly factors, including the small nucleolar RNA snR30. Through interacting with the unprocessed precursor ribosomal RNA (pre-rRNA), snR30 is essential for the maturation of pre-rRNA forming the small ribosomal subunit. To characterize snR30’s interaction network, an in vitro approach was used to quantify the affinity of snR30 to the core H/ACA protein complex and the PIN endonuclease Utp23 revealing very tight interactions with affinities in the sub- and low-nanomolar ranges, respectively. Furthermore, the snR30 complex binds tightly to its primary binding site in ribosomal RNA called the C1 site, but only weakly to other predicted interaction sites. Utp23 binds tightly to its predicted interaction site in ribosomal RNA only if adjacent rRNA elements are also present. In conclusion, these findings serve as foundational work to further explore the mechanisms of snR30 during maturation of rRNA.

The creation of eukaryotic ribosomes is a complex process involving hundreds of assembly factors, including the small nucleolar RNA snR30. Through interacting with the unprocessed precursor ribosomal RNA (pre-rRNA), snR30 is essential for the maturation of pre-rRNA forming the small ribosomal subunit. To characterize snR30’s interaction network, an in vitro approach was used to quantify the affinity of snR30 to the core H/ACA protein complex and the PIN endonuclease Utp23 revealing very tight interactions with affinities in the sub- and low-nanomolar ranges, respectively. Furthermore, the snR30 complex binds tightly to its primary binding site in ribosomal RNA called the C1 site, but only weakly to other predicted interaction sites. Utp23 binds tightly to its predicted interaction site in ribosomal RNA only if adjacent rRNA elements are also present. In conclusion, these findings serve as foundational work to further explore the mechanisms of snR30 during maturation of rRNA.

Antibiotic resistance is becoming an increasing global health concern. The bacterial protein synthesis machinery, including the ribosome and its associated factors, are the target of over half of the clinically relevant antibiotics currently in use, highlighting the importance of cellular protein production as antibiotic target. The ribosome associated factors HflX and YchF are members of the GTPase superfamily. Their cellular functions are only poorly understood. The overarching goal of this thesis was to determine the location where HflX and YchF bind on the bacterial ribosome. Data presented here confirm that YchF interacts with the ribosomal A-site, while HflX is unique within the GTPase family being able to bind to both the ribosomal A-site and E-site. From this data and subsequent biochemical and biophysical studies, a mechanism for both HflX and YchF function during protein synthesis, specifically under stress conditions, is proposed.

Antibiotic resistance is becoming an increasing global health concern. The bacterial protein synthesis machinery, including the ribosome and its associated factors, are the target of over half of the clinically relevant antibiotics currently in use, highlighting the importance of cellular protein production as antibiotic target. The ribosome associated factors HflX and YchF are members of the GTPase superfamily. Their cellular functions are only poorly understood. The overarching goal of this thesis was to determine the location where HflX and YchF bind on the bacterial ribosome. Data presented here confirm that YchF interacts with the ribosomal A-site, while HflX is unique within the GTPase family being able to bind to both the ribosomal A-site and E-site. From this data and subsequent biochemical and biophysical studies, a mechanism for both HflX and YchF function during protein synthesis, specifically under stress conditions, is proposed.

The creation of eukaryotic ribosomes is a complex process involving hundreds of assembly factors, including the small nucleolar RNA snR30. Through interacting with the unprocessed precursor ribosomal RNA (pre-rRNA), snR30 is essential for the maturation of pre-rRNA forming the small ribosomal subunit. To characterize snR30’s interaction network, an in vitro approach was used to quantify the affinity of snR30 to the core H/ACA protein complex and the PIN endonuclease Utp23 revealing very tight interactions with affinities in the sub- and low-nanomolar ranges, respectively. Furthermore, the snR30 complex binds tightly to its primary binding site in ribosomal RNA called the C1 site, but only weakly to other predicted interaction sites. Utp23 binds tightly to its predicted interaction site in ribosomal RNA only if adjacent rRNA elements are also present. In conclusion, these findings serve as foundational work to further explore the mechanisms of snR30 during maturation of rRNA.

The creation of eukaryotic ribosomes is a complex process involving hundreds of assembly factors, including the small nucleolar RNA snR30. Through interacting with the unprocessed precursor ribosomal RNA (pre-rRNA), snR30 is essential for the maturation of pre-rRNA forming the small ribosomal subunit. To characterize snR30’s interaction network, an in vitro approach was used to quantify the affinity of snR30 to the core H/ACA protein complex and the PIN endonuclease Utp23 revealing very tight interactions with affinities in the sub- and low-nanomolar ranges, respectively. Furthermore, the snR30 complex binds tightly to its primary binding site in ribosomal RNA called the C1 site, but only weakly to other predicted interaction sites. Utp23 binds tightly to its predicted interaction site in ribosomal RNA only if adjacent rRNA elements are also present. In conclusion, these findings serve as foundational work to further explore the mechanisms of snR30 during maturation of rRNA.

Antibiotic resistance is becoming an increasing global health concern. The bacterial protein synthesis machinery, including the ribosome and its associated factors, are the target of over half of the clinically relevant antibiotics currently in use, highlighting the importance of cellular protein production as antibiotic target. The ribosome associated factors HflX and YchF are members of the GTPase superfamily. Their cellular functions are only poorly understood. The overarching goal of this thesis was to determine the location where HflX and YchF bind on the bacterial ribosome. Data presented here confirm that YchF interacts with the ribosomal A-site, while HflX is unique within the GTPase family being able to bind to both the ribosomal A-site and E-site. From this data and subsequent biochemical and biophysical studies, a mechanism for both HflX and YchF function during protein synthesis, specifically under stress conditions, is proposed.

Antibiotic resistance is becoming an increasing global health concern. The bacterial protein synthesis machinery, including the ribosome and its associated factors, are the target of over half of the clinically relevant antibiotics currently in use, highlighting the importance of cellular protein production as antibiotic target. The ribosome associated factors HflX and YchF are members of the GTPase superfamily. Their cellular functions are only poorly understood. The overarching goal of this thesis was to determine the location where HflX and YchF bind on the bacterial ribosome. Data presented here confirm that YchF interacts with the ribosomal A-site, while HflX is unique within the GTPase family being able to bind to both the ribosomal A-site and E-site. From this data and subsequent biochemical and biophysical studies, a mechanism for both HflX and YchF function during protein synthesis, specifically under stress conditions, is proposed.

The creation of eukaryotic ribosomes is a complex process involving hundreds of assembly factors, including the small nucleolar RNA snR30. Through interacting with the unprocessed precursor ribosomal RNA (pre-rRNA), snR30 is essential for the maturation of pre-rRNA forming the small ribosomal subunit. To characterize snR30’s interaction network, an in vitro approach was used to quantify the affinity of snR30 to the core H/ACA protein complex and the PIN endonuclease Utp23 revealing very tight interactions with affinities in the sub- and low-nanomolar ranges, respectively. Furthermore, the snR30 complex binds tightly to its primary binding site in ribosomal RNA called the C1 site, but only weakly to other predicted interaction sites. Utp23 binds tightly to its predicted interaction site in ribosomal RNA only if adjacent rRNA elements are also present. In conclusion, these findings serve as foundational work to further explore the mechanisms of snR30 during maturation of rRNA.

The creation of eukaryotic ribosomes is a complex process involving hundreds of assembly factors, including the small nucleolar RNA snR30. Through interacting with the unprocessed precursor ribosomal RNA (pre-rRNA), snR30 is essential for the maturation of pre-rRNA forming the small ribosomal subunit. To characterize snR30’s interaction network, an in vitro approach was used to quantify the affinity of snR30 to the core H/ACA protein complex and the PIN endonuclease Utp23 revealing very tight interactions with affinities in the sub- and low-nanomolar ranges, respectively. Furthermore, the snR30 complex binds tightly to its primary binding site in ribosomal RNA called the C1 site, but only weakly to other predicted interaction sites. Utp23 binds tightly to its predicted interaction site in ribosomal RNA only if adjacent rRNA elements are also present. In conclusion, these findings serve as foundational work to further explore the mechanisms of snR30 during maturation of rRNA.

Antibiotic resistance is becoming an increasing global health concern. The bacterial protein synthesis machinery, including the ribosome and its associated factors, are the target of over half of the clinically relevant antibiotics currently in use, highlighting the importance of cellular protein production as antibiotic target. The ribosome associated factors HflX and YchF are members of the GTPase superfamily. Their cellular functions are only poorly understood. The overarching goal of this thesis was to determine the location where HflX and YchF bind on the bacterial ribosome. Data presented here confirm that YchF interacts with the ribosomal A-site, while HflX is unique within the GTPase family being able to bind to both the ribosomal A-site and E-site. From this data and subsequent biochemical and biophysical studies, a mechanism for both HflX and YchF function during protein synthesis, specifically under stress conditions, is proposed.

The creation of eukaryotic ribosomes is a complex process involving hundreds of assembly factors, including the small nucleolar RNA snR30. Through interacting with the unprocessed precursor ribosomal RNA (pre-rRNA), snR30 is essential for the maturation of pre-rRNA forming the small ribosomal subunit. To characterize snR30’s interaction network, an in vitro approach was used to quantify the affinity of snR30 to the core H/ACA protein complex and the PIN endonuclease Utp23 revealing very tight interactions with affinities in the sub- and low-nanomolar ranges, respectively. Furthermore, the snR30 complex binds tightly to its primary binding site in ribosomal RNA called the C1 site, but only weakly to other predicted interaction sites. Utp23 binds tightly to its predicted interaction site in ribosomal RNA only if adjacent rRNA elements are also present. In conclusion, these findings serve as foundational work to further explore the mechanisms of snR30 during maturation of rRNA.

Ribosome biogenesis is a crucial yet poorly understood and complex process in all cells. To date, most studies on eukaryotic ribosome biogenesis have relied on yeast genetics and whole cell analysis of ribosomal RNA processing. An early and critical step in ribosome biogenesis is the post-transcriptional modification of rRNA. Pseudouridylation is the most frequently occurring modification. Pseudouridylation is catalyzed by H/ACA small nucleolar ribonucleoproteins (snoRNPs) which are one of the two major classes of snoRNPs found within eukaryotes and archaea. H/ACA snoRNPs consist of four conserved core proteins Cbf5, Gar1, Nop10, and Nhp2 (eukaryotes), and a substrate specific H/ACA snoRNA. Mutations causing the rare inherited disease Dyskeratosis congenita are found in CBF5, NOP10, and NHP2. Here I report the purification of H/ACA protein Cbf5 in the presence of detergents. Additionally, I report initial in vitro RNA binding studies using Nhp2 and the snoRNA snR34 as well as the effects of Dyskeratosis congenita substitutions within Nhp2 on this interaction.

Ribosome biogenesis is a crucial yet poorly understood and complex process in all cells. To date, most studies on eukaryotic ribosome biogenesis have relied on yeast genetics and whole cell analysis of ribosomal RNA processing. An early and critical step in ribosome biogenesis is the post-transcriptional modification of rRNA. Pseudouridylation is the most frequently occurring modification. Pseudouridylation is catalyzed by H/ACA small nucleolar ribonucleoproteins (snoRNPs) which are one of the two major classes of snoRNPs found within eukaryotes and archaea. H/ACA snoRNPs consist of four conserved core proteins Cbf5, Gar1, Nop10, and Nhp2 (eukaryotes), and a substrate specific H/ACA snoRNA. Mutations causing the rare inherited disease Dyskeratosis congenita are found in CBF5, NOP10, and NHP2. Here I report the purification of H/ACA protein Cbf5 in the presence of detergents. Additionally, I report initial in vitro RNA binding studies using Nhp2 and the snoRNA snR34 as well as the effects of Dyskeratosis congenita substitutions within Nhp2 on this interaction.

Ribosome biogenesis is a crucial yet poorly understood and complex process in all cells. To date, most studies on eukaryotic ribosome biogenesis have relied on yeast genetics and whole cell analysis of ribosomal RNA processing. An early and critical step in ribosome biogenesis is the post-transcriptional modification of rRNA. Pseudouridylation is the most frequently occurring modification. Pseudouridylation is catalyzed by H/ACA small nucleolar ribonucleoproteins (snoRNPs) which are one of the two major classes of snoRNPs found within eukaryotes and archaea. H/ACA snoRNPs consist of four conserved core proteins Cbf5, Gar1, Nop10, and Nhp2 (eukaryotes), and a substrate specific H/ACA snoRNA. Mutations causing the rare inherited disease Dyskeratosis congenita are found in CBF5, NOP10, and NHP2. Here I report the purification of H/ACA protein Cbf5 in the presence of detergents. Additionally, I report initial in vitro RNA binding studies using Nhp2 and the snoRNA snR34 as well as the effects of Dyskeratosis congenita substitutions within Nhp2 on this interaction.

Ribosome biogenesis is a crucial yet poorly understood and complex process in all cells. To date, most studies on eukaryotic ribosome biogenesis have relied on yeast genetics and whole cell analysis of ribosomal RNA processing. An early and critical step in ribosome biogenesis is the post-transcriptional modification of rRNA. Pseudouridylation is the most frequently occurring modification. Pseudouridylation is catalyzed by H/ACA small nucleolar ribonucleoproteins (snoRNPs) which are one of the two major classes of snoRNPs found within eukaryotes and archaea. H/ACA snoRNPs consist of four conserved core proteins Cbf5, Gar1, Nop10, and Nhp2 (eukaryotes), and a substrate specific H/ACA snoRNA. Mutations causing the rare inherited disease Dyskeratosis congenita are found in CBF5, NOP10, and NHP2. Here I report the purification of H/ACA protein Cbf5 in the presence of detergents. Additionally, I report initial in vitro RNA binding studies using Nhp2 and the snoRNA snR34 as well as the effects of Dyskeratosis congenita substitutions within Nhp2 on this interaction.

Ribosome biogenesis is a crucial yet poorly understood and complex process in all cells. To date, most studies on eukaryotic ribosome biogenesis have relied on yeast genetics and whole cell analysis of ribosomal RNA processing. An early and critical step in ribosome biogenesis is the post-transcriptional modification of rRNA. Pseudouridylation is the most frequently occurring modification. Pseudouridylation is catalyzed by H/ACA small nucleolar ribonucleoproteins (snoRNPs) which are one of the two major classes of snoRNPs found within eukaryotes and archaea. H/ACA snoRNPs consist of four conserved core proteins Cbf5, Gar1, Nop10, and Nhp2 (eukaryotes), and a substrate specific H/ACA snoRNA. Mutations causing the rare inherited disease Dyskeratosis congenita are found in CBF5, NOP10, and NHP2. Here I report the purification of H/ACA protein Cbf5 in the presence of detergents. Additionally, I report initial in vitro RNA binding studies using Nhp2 and the snoRNA snR34 as well as the effects of Dyskeratosis congenita substitutions within Nhp2 on this interaction.

Ribosome biogenesis is a crucial yet poorly understood and complex process in all cells. To date, most studies on eukaryotic ribosome biogenesis have relied on yeast genetics and whole cell analysis of ribosomal RNA processing. An early and critical step in ribosome biogenesis is the post-transcriptional modification of rRNA. Pseudouridylation is the most frequently occurring modification. Pseudouridylation is catalyzed by H/ACA small nucleolar ribonucleoproteins (snoRNPs) which are one of the two major classes of snoRNPs found within eukaryotes and archaea. H/ACA snoRNPs consist of four conserved core proteins Cbf5, Gar1, Nop10, and Nhp2 (eukaryotes), and a substrate specific H/ACA snoRNA. Mutations causing the rare inherited disease Dyskeratosis congenita are found in CBF5, NOP10, and NHP2. Here I report the purification of H/ACA protein Cbf5 in the presence of detergents. Additionally, I report initial in vitro RNA binding studies using Nhp2 and the snoRNA snR34 as well as the effects of Dyskeratosis congenita substitutions within Nhp2 on this interaction.

Ribosome biogenesis is a crucial yet poorly understood and complex process in all cells. To date, most studies on eukaryotic ribosome biogenesis have relied on yeast genetics and whole cell analysis of ribosomal RNA processing. An early and critical step in ribosome biogenesis is the post-transcriptional modification of rRNA. Pseudouridylation is the most frequently occurring modification. Pseudouridylation is catalyzed by H/ACA small nucleolar ribonucleoproteins (snoRNPs) which are one of the two major classes of snoRNPs found within eukaryotes and archaea. H/ACA snoRNPs consist of four conserved core proteins Cbf5, Gar1, Nop10, and Nhp2 (eukaryotes), and a substrate specific H/ACA snoRNA. Mutations causing the rare inherited disease Dyskeratosis congenita are found in CBF5, NOP10, and NHP2. Here I report the purification of H/ACA protein Cbf5 in the presence of detergents. Additionally, I report initial in vitro RNA binding studies using Nhp2 and the snoRNA snR34 as well as the effects of Dyskeratosis congenita substitutions within Nhp2 on this interaction.

Ribosome biogenesis is a crucial yet poorly understood and complex process in all cells. To date, most studies on eukaryotic ribosome biogenesis have relied on yeast genetics and whole cell analysis of ribosomal RNA processing. An early and critical step in ribosome biogenesis is the post-transcriptional modification of rRNA. Pseudouridylation is the most frequently occurring modification. Pseudouridylation is catalyzed by H/ACA small nucleolar ribonucleoproteins (snoRNPs) which are one of the two major classes of snoRNPs found within eukaryotes and archaea. H/ACA snoRNPs consist of four conserved core proteins Cbf5, Gar1, Nop10, and Nhp2 (eukaryotes), and a substrate specific H/ACA snoRNA. Mutations causing the rare inherited disease Dyskeratosis congenita are found in CBF5, NOP10, and NHP2. Here I report the purification of H/ACA protein Cbf5 in the presence of detergents. Additionally, I report initial in vitro RNA binding studies using Nhp2 and the snoRNA snR34 as well as the effects of Dyskeratosis congenita substitutions within Nhp2 on this interaction.

Ribosome biogenesis is a crucial yet poorly understood and complex process in all cells. To date, most studies on eukaryotic ribosome biogenesis have relied on yeast genetics and whole cell analysis of ribosomal RNA processing. An early and critical step in ribosome biogenesis is the post-transcriptional modification of rRNA. Pseudouridylation is the most frequently occurring modification. Pseudouridylation is catalyzed by H/ACA small nucleolar ribonucleoproteins (snoRNPs) which are one of the two major classes of snoRNPs found within eukaryotes and archaea. H/ACA snoRNPs consist of four conserved core proteins Cbf5, Gar1, Nop10, and Nhp2 (eukaryotes), and a substrate specific H/ACA snoRNA. Mutations causing the rare inherited disease Dyskeratosis congenita are found in CBF5, NOP10, and NHP2. Here I report the purification of H/ACA protein Cbf5 in the presence of detergents. Additionally, I report initial in vitro RNA binding studies using Nhp2 and the snoRNA snR34 as well as the effects of Dyskeratosis congenita substitutions within Nhp2 on this interaction.

Ribosome biogenesis is a crucial yet poorly understood and complex process in all cells. To date, most studies on eukaryotic ribosome biogenesis have relied on yeast genetics and whole cell analysis of ribosomal RNA processing. An early and critical step in ribosome biogenesis is the post-transcriptional modification of rRNA. Pseudouridylation is the most frequently occurring modification. Pseudouridylation is catalyzed by H/ACA small nucleolar ribonucleoproteins (snoRNPs) which are one of the two major classes of snoRNPs found within eukaryotes and archaea. H/ACA snoRNPs consist of four conserved core proteins Cbf5, Gar1, Nop10, and Nhp2 (eukaryotes), and a substrate specific H/ACA snoRNA. Mutations causing the rare inherited disease Dyskeratosis congenita are found in CBF5, NOP10, and NHP2. Here I report the purification of H/ACA protein Cbf5 in the presence of detergents. Additionally, I report initial in vitro RNA binding studies using Nhp2 and the snoRNA snR34 as well as the effects of Dyskeratosis congenita substitutions within Nhp2 on this interaction.