Investigation into the Mechanism of the
Protein Subunit of RNase P
Hao-Hsun Chang
Yoshio Ikeda
Wei Liu
Xudong Zhang
Zhongzhou Zheng
Ohio State Biochemistry Program
The Ohio State University
March 5th, 2004
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Chang, Ikeda, Liu, Zhang, and Zheng
Background and Significance
RNase P (EC 3.1.26.5) is an endonuclease that cleaves pre-tRNA at 5’ leader
region in the maturation process of tRNA. It is a ribonucleoprotein (RNP) complex
containing a single RNA subunit and a protein subunit whose composition varies
among phylogenetic domains. The remarkable aspect of RNase P is that it is the RNA
moiety that catalyzes the cleavage, rather than the protein subunit. In this sense,
RNase P is a ribozyme.
However, the protein cofactor is essential for the growth of E. coli.
1
The
elucidation of the functional role of the protein subunit can yield the detailed
information on the overall mechanisms for the RNase P catalysis and paves the way
for the studies on the properties and the functions of the protein subunits in other
ribonucleoprotein (RNP) complexes.
At first, researchers suspected that the main role of the protein subunit is to
promote P-RNA folding into a catalytically active form, as Saccharomyces cerevisiae
Group I B15 intron requires CBP2 protein to stabilize the tertiary structure of its
active site to enhance splicing. But later, it was found that the E. coli and B. subtilis
RNase P RNAs are fully folded at equilibrium in 6 mM Mg2+ and that the presence of
P Protein only has modest effects on the global folding of P RNA.
2, 3, 4
Moreover, the
RNase P RNA subunit alone is sufficient for the catalysis of pre-tRNA cleavage in
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vitro.
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Chang, Ikeda, Liu, Zhang, and Zheng
However, compared to the holoenzyme-catalyzed reaction, the P
RNA-catalyzed reaction requires high concentrations of monovalent and divalent
cations (preferably Mg2+).
6
A group of researchers found that Mg2+ enhances the
affinity of RNase P RNA for pre-tRNA and mature tRNA, and stabilizes the transition
state for pre-tRNA cleavage.
7
On the other hand, it has also been demonstrated that
the protein subunit has multiple effects on the kinetics of pre-tRNA hydrolysis and it
increases the affinity of pre-tRNA substrate by a factor of 104 while having a more
modest effect on the affinity of mature tRNA.
8
These two parallel phenomena
prompted scientists to hypothesize that the functional role of RNase P protein subunit
is to increase the affinity of RNase P for Mg2+ so that Mg2+ can be recruited more
efficiently to exert their positive effects on substrate binding and chemical cleavage,
but not on mature tRNA binding.
Several experiments were conducted to test this hypothesis, as detailed below: 9
1. Magnesium ions bind to P RNA in two ways. More than 100 magnesium ions
bind nonspecifically through electrostatic forces to the RNA polyanion. Several
additional magnesium ions bind to some specific sites to exert functions. It was shown
that the addition of the protein subunit does not affect the electrostatic binding of
magnesium ions to RNA polyanion (data not shown).
2. Effects of magnesium on single turnover kinetics
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Chang, Ikeda, Liu, Zhang, and Zheng
Single-turnover is achieved by using excess enzyme molecules. Previous studies
7
have revealed that the single-turnover rate constant k2 is dependent on Mg2+
concentration, and can be analyzed with Hill equation: 11
Kobs=kmax([Mg2+]αH/K1/2αH)/(1+[Mg2+]αH/K1/2αH)
(1)
K1/2 is the magnesium concentration at which k2=1/2×k2,max , reflecting the
affinity of P RNA for Mg2+. αH is the Hill coefficient, which reflects the cooperativity
of Mg2+ binding.
Equation (1) has a linearized form:
Log(f/1-f)=αHlog[Mg2+]+bk(K1/2)
(2)
in which f=k2/k2,max; bk(K1/2) is the interception, which is a function of K1/2.
Previously, by analyzing the Mg2+ dependence for the cleavage of pre-tRNA
catalyzed by P RNA at high salt concentration, the results that αH=1-3 and K1/2＞＝20
mM were derived. If the protein subunit is added, k2 is unaffected under the MgCl2
concentration varying from 4 to 20 mM (MgCl2 concentration cannot be lower than 4
mM because that will influence the folding of P RNA), which indicates that with the
protein subunit K1/2 is less than 4 mM. Consequently, the protein subunit enhances the
affinity for magnesium ions. 7
To measure the Mg2+ dependence of the substrate binding affinity KDpre-tRNA, the
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Chang, Ikeda, Liu, Zhang, and Zheng
substrates with short leader sequence (1 nucleotide) and various reaction conditions
were used to alter the kinetic mechanism from the previous “two consecutive
irreversible first-order reaction” to a rapid equilibrium mechanism. The dependence of
KD1-tRNA on Mg2+ can also be analyzed using the linearized form of Hill equation
(equation (2)):
Log(f/1-f)=αHlog[Mg2+]+bD(K1/2)
(2)
in which, f=KD,min1-tRNA/KD1-tRNA; bD(K1/2) is the interception, a function of K1/2.
O: k2 at pH 6.0
●: k2 at pH 5.2
□: KD1-tRNA at pH 5.2
The results showed that, for both k2 and KD1-tRNA, Hill coefficient is around 4.
This, together with other observations, made the researchers conclude that at least
four magnesium ions of the same set bind cooperatively to RNase P at the presence of
the protein subunit, which serves both to stabilize the binding of pre-tRNA and to
accelerate the cleavage process.
3. Binding of mature tRNA to RNase P
Bound and free mature tRNAAsp were separated by gel filtration. KDtRNA was
calculated using the following equation:
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Chang, Ikeda, Liu, Zhang, and Zheng
[E˙L]/[L]total=1/(1+KD/[E]total)
(3)
○:RNase P RNA
▲:Holoenzyme
The affinity of the holoenzyme for the mature tRNA is only modestly enhanced.
The Hill plot of the dependence of KDtRNA on the MgCl2 concentration further
demonstrates that the protein subunit has little effect on the affinity or the
cooperativity of Mg2+ which enhances the mature tRNA binding to RNase P.
(αH=3.4-0.2 and K1/2=78-12 for RNase P RNA; αH=2.7-0.4 and K1/2=53-13 for the
holoenzyme)
The data above, together with the finding that the interaction between P protein
and pre-tRNA 5’ leader region is important for the function of the protein subunit
and some other structural analysis
13
12
, lead the scientists to a hypothetical conclusion
that the interaction between P protein and pre-tRNA 5’ leader region induces a
structural change in RNase P RNA providing at least four high-affinity specific Mg2+
binding sites. The magnesium ions recruited in this way help to increase the affinity of
RNase P for the pre-tRNA substrate, and/or to stabilize the transition state to
accelerate the cleavage.
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Chang, Ikeda, Liu, Zhang, and Zheng
Specific Aims and Approaches
Our group wants to look into the mechanism how the structural change induced
by P protein in RNA P provides the high-affinity specific Mg2+ binding sites. We are
focused on how P RNA provides the specific Mg2+ binding sites to bind Mg2+ to
stabilize the transition state of the cleavage reaction. According to a proposed general
two-metal mechanism 14 illustrated below, two divalent metal ions are coordinated by
oxygen. These ions serve to delocalize the accumulation of the negative charges in the
transition state, thereby stabilizing the transition state and facilitating the catalysis. We
hypothesize that the structural change of P RNA induced by the interaction of P
protein and pre-tRNA forms two high-affinity Mg2+ binding sites near the active site
so that two magnesium ions are positioned at these two sites where they are
coordinated.
To examine this hypothesis, we plan to use NMR first to monitor any structural
change of RNase P RNA in the proximity of the active site. If any such structural
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Chang, Ikeda, Liu, Zhang, and Zheng
change is found, we will further study the nucleotides involved in the structural
change and look for some candidate sites for Mg2+ binding, e.g. sugar hydroxyl
groups, base ring nitrogen atoms (e.g. N7 of Guanine) and exocyclic base keto groups
(e.g. O2 of Cytosine). The general feature of these sites is that they possess lone-pair
electrons to bind the orbital of metal ions. We can make some modification, such as
methylation, on these candidate nitrogen and oxygen atoms, which will attenuate the
lone-pair electron cloud and provide some steric hindrance for the metal ion binding.
If any of the candidate sites is indeed involved in Mg2+ binding, the modification on
the site will definitely decrease the cleavage efficiency. Through this way, we can
obtain some general knowledge on how specific magnesium binding sites are formed
upon structural change, which will push forward our understanding on how
interaction between P protein and pre-tRNA triggers this event.
Another question that our group would like to address is whether P protein plays
any role other than increasing the Mg2+ affinity. We are interested in this question
because P protein in B. subtilis has a positive though modest effect on the rate
constant for the phosphodiester cleavage of pre-tRNAAsp catalyzed by RNase P at the
saturating concentration of Mg2+ and substrates. It is interesting to hypothesize that
the positively charged residue(s) (Lys or Arg) in P protein, placed in the proximity of
the cleavage site, may also help to neutralize the negative charge pool formed in the
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Chang, Ikeda, Liu, Zhang, and Zheng
transition state and thus facilitate the cleavage.
With the mutation at some positively charged residues near cleavage site to Ala,
we expect to see no enhancement in the rate constant upon adding the mutant P
protein at the saturating Mg2+ concentration. Dr. Gopalan’s research group has
mutated some conserved positively charged residues in P protein, including the
residues in the highly conserved RNR motif, which has been demonstrated to mainly
interact with the P3 and P4 helices in the catalytic domain of RNase P RNA 15, into
Ala residues to observe the consequence. Their result is somewhat surprising: all the
function of P protein was totally abolished for the mutant P protein 16. This result can
be explained by presuming that the conserved positive charged residues are essential
for P protein binding to P RNA. This result also prompts our group to hypothesize that
because the binding site happens to be located near the catalysis active site, the
positive charged residues coincidentally help to relieve the negative charge
accumulation in the transition state. In other words, the conserved positive charged
residues are not “born” to stabilize the transition state, which accounts for the modest
rate acceleration observed previously.
To verify this hypothesis, we are considering mutating more residues into
positively charged residues near the binding site and near the catalytically active site.
As long as these mutations do not interfere with the structure and stability of the
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Chang, Ikeda, Liu, Zhang, and Zheng
protein, we would expect to see greater cleavage rate enhancement for the mutant P
protein compared to the wild type molecule.
In summary, our group makes the best of our current knowledge to investigate
into the detailed function role of the RNase P protein subunit on both the
Mg2+-dependent and Mg2+-independent mechanisms. Although the complete reveal of
the function of the protein subunit in RNase P is still a long way to go, our study will
certainly make a great progress towards the further understanding.
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