Identification of binding sites of TEFM with mitochondrial RNA polymerase

James Graber

Rowan University Summer Undergraduate Research Experience 2015

Department of Cell Biology

Dr. Dmitry Temiakov

1

Abstract

Effective regulation over transcription and replication of the mitochondrial

genome is critical during spermatogenesis and the early stages of embryogenesis. In these

stages of cell development, mitochondria increases respiration and adenosine

triphosphate production without needing to replicate its genome. Transcription elongation

factor of mitochondria (TEFM), in concert with mitochondrial RNA polymerase

(mtRNAP), behaves as a molecular switch between replication and transcription in

mitochondria and may be responsible for increasing transcription rates during cell

development.1 We substituted sites along the N-terminal domain of TEFM with the

amber codon and used nonsense suppressors to incorporate the unnatural amino acid p-

benzoyl-L-phenylalanine (Bpa), a photoreactive crosslinker. The TEFM-Bpa mutant

underwent crosslinking with mtRNAP in an effort to identify binding sites. Identification

of binding sites will allow complete structural mapping of TEFM and mtRNAP

interaction. From there a molecular mechanism explaining TEFM’s functionality can be

proposed.

2

Introduction

TEFM behaves as a molecular switch between replication and transcription, two

mutually exclusive events in human mitochondrial DNA (mtDNA). In the absence of

TEFM, transcription of mtDNA terminates prematurely downstream of the light-strand

promotor (LSP) at the conserved sequence block II (CSBII). This event generates a

replication primer near the origin of replication of the heavy strand, thus permitting

initiation of replication. In the presence of TEFM, an elongation complex is formed

between human mitochondrial RNA polymerase (mtRNAP), nascent RNA transcript,

mtDNA (region 202-481), and TEFM. When TEFM is bound to mtRNAP, mtRNAP

efficiently transcribes through CSBII, producing long transcripts. Thus TEFM regulation

enables cells to control both mtDNA copy number and transcription rates.1

In an effort to better understand how TEFM interacts with mtRNAP to prevent

premature termination at the CSBII, we sought to map binding sites between TEFM and

the elongation complex by site-directed mutagenesis of an unnatural amino acid and

photocrosslinker, Bpa. We substituted codons encoding for TEFM amino acid residues

K55, K63, S72, R89, Q110, and K122 with the amber nonsense codon (TAG).

Substitution of R89 with photocrosslinker Bpa was accomplished using nonsense

suppressors, a system described by Young and colleagues.2 Once TEFM-R89Bpa was

purified, a series of crosslinking experiments were performed by assembling the

elongation complexes with the TEFM mutant. The experiment was followed by a

transcription assay to observe whether the R89Bpa mutation obstructs TEFM’s anti-

termination functionality.

3

Materials and Methods

The TEFM gene was cloned onto pET-21b plasmid vector (ampicillin resistance,

IPTG-inducible) in mature form (residues 36-360) with C-terminal His-6 tag. Addition of

synthetic oligonucleotide primers followed by polymerase chain reaction (PCR)

introduced the amber (TAG) mutation into the TEFM gene by substitution. Exposure to

Dpn I endonuclease, which targets methylated and hemimethylated DNA, for 1 hour at

37ᵒC selected for vectors containing the mutation. Vector encoding modified TEFM gene

was amplified by plating transformed XL10-Gold ultracompetent cells with ampicillin

and selecting single colony for overnight culture at 37ᵒC. The vector was then isolated by

miniprep. BLR (DE3) electrocompetent cells were transformed with mutated pET-21b

and pEVOL-Bpa vectors via electroporation. pEVOL-Bpa (chloramphenicol resistance,

arabinose-inducible) encodes for an aminoacyl-tRNA synthetase (aaRS) / suppressor

tRNA pair which incorporates the unnatural amino acid and photoreactive crosslinker, p-

benzoyl-L-phenylalanine (Bpa), in response to the amber nonsense codon during

translation.2 BLR (DE3) cells were then selected for via plating with ampicillin and

chloramphenicol. Selected cells were cultured at 37ᵒC to an optical density at 600 nm of

0.4 units in the presence of 1 mM Bpa. Cultures were cooled to 16ᵒC and induced with

0.1 mM IPTG and 0.02% arabinose for 20 hours. Cells were then disrupted by sonication.

TEFM-Bpa mutant was purified using Ni-NTA Agarose chromatography, heparin-

sepharose chromatography, and gel filtration (in that order).

Templates used in transcription assays contained the region 202-481 of human

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mitochondrial DNA (hmtDNA) with a deletion of 71 base pairs between the LSP and

CSBII (G6AG8). Transcription reactions contained DNA template (50 nM), mtRNAP

(150 nM), TFAM (250 nM), TFB2M (150 nM), TEFM (300-1200 nM), ATP/GTP/CTP

(3 mM), UTP (0.1 mM), and radioactive label [α-32P] UTP in transcription buffer (20 mM

Tris pH=8.0, 10 mM MgCl2, 10 mM, β-mercaptoethanol). TFAM and TFB2M are

required for effective initiation of transcription with mtRNAP in mtDNA.3 Reactions

were carried out at 35ᵒC for 30 minutes and stopped by addition of an equal volume of

95% formamide/ 0.05M EDTA solution. Products were resolved by 20% PAGE

containing 6 M urea and visualized by phosphorimagining.

Three scaffolds for crosslinking (RNA/DNA, 5’ radiolabeled-RNA/DNA,

RNA/5’ radiolabeled-DNA) were prepared by exposure to T4 polynucleotide kinase

(PNK) in the presence of radioactive label γ-32P[ATP] for 30 minutes at 37ᵒC in PNK

Buffer A. Then PNK was inactivated by incubation at 95ᵒC for 3 minutes. RNA and DNA

oligonucleotides were annealed at 95ᵒC for 5 minutes, followed by gradual temperature

decrease to 25ᵒC. mtRNAP was radiolabeled by exposure to protein kinase A (PKA) in

the presence of γ-32P[ATP] for 30 minutes at room temperature in PK Buffer. Elongation

complexes were assembled by UV-irradiation (312 nm) of 0.2 µM scaffold, 0.2 µM

mtRNAP, and 2 µM TEFM for 30 minutes at room temperature. Bpa, when UV-

irradiated, covalently crosslinks to a second species within 4 angstrom. Products were

resolved using 4-12% SDS-PAGE and visualized by phosphorimagining.

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Results

TEFM-R89Bpa did not crosslink with mtRNAP or scaffold (Fig. 1). Furthermore,

substitution of R89 with Bpa in TEFM did not have detrimental effects on TEFM’s anti-

termination activity (Fig. 2). Juxtaposition of data suggests that amino acid residue R89

does not play a role in TEFM’s binding to the elongation complex or TEFM’s anti-

termination activity.

Lane

sReaction

1 radiolabeled-mtRNAP / TEFM-WT

2 radiolabeled-mtRNAP / TEFM-R89Bpa

3 radiolabeled-mtRNAP / scaffold / TEFM-R89Bpa

4 mtRNAP / radiolabeled-RNA scaffold / TEFM-WT

5 mtRNAP / radiolabeled-RNA scaffold / TEFM-R89Bpa

6 mtRNAP / radiolabeled-DNA scaffold / TEFM-WT

7 mtRNAP / radiolabeled-DNA scaffold / TEFM-R89Bpa

8 radiolabeled-RNA scaffold / TEFM-R89Bpa

9 radiolabeled-DNA scaffold / TEFM-R89Bpa

Figure 1: Phosphorimagining of crosslinking experiment. TEFM-R89Bpa fails to

crosslink with mtRNAP and scaffold. Results are compared to wild-type (WT) TEFM.

6

WT-TEFM TEFM-R89BpaTEFM (µM) 0.3 0.6 0.3 0.6 1.2

Run-off

Termination at CSBII

Figure 2: Phosphorimagining of transcription assay. R89Bpa mutation does not

obstruct TEFM’s anti-termination activity. Results are compared to wild-type (WT)

TEFM.

7

Discussion

Our in vitro data shows that amino acid residue R89 of TEFM is not a binding site

in the elongation complex. Moreover, R89 has no significant function in preventing

termination at CSBII. We also introduced amber nonsense (TAG) mutations to codons

encoding charged amino acid residues along the N-terminal domain of TEFM: K55, K63,

S72, Q110, and K122. Further in vitro analysis of these residues using unnatural amino

acid mutagenesis of photocrosslinker Bpa should be explored so that the molecular

mechanism of TEFM’s anti-termination activity can be better understood. Furthermore,

in considering the significance of mitochondrial genetics in cell development,

mitochondrial diseases, neurological disorders, and senescence, an understanding of the

regulation system for the molecular switch TEFM in living cells could prove beneficial in

medicine.

8

References

1. K. Agaronyan, Y. I. Morozov, M. Anikin, and D. Temiakov, Replication-

transcription switch in human mitochondria, Science. 30 January

2015: 347 (6221), 548-551. doi:10.1126/science.aaa0986.

2. T. S. Young, I. Ahmad, J. A. Yin, and P. G. Schultz, An Enhanced System for

Unnatural Amino Acid Mutagenesis in E. coli, Journal of Molecular Biology,

Volume 395, Issue 2, 15 January 2010, Pages 361-374, ISSN 0022-2836,

http://dx.doi.org/10.1016/j.jmb.2009.10.030.

(http://www.sciencedirect.com/science/article/pii/S0022283609012704)

3. Y. I. Morozov, K. Agaronyan, A. C. M. Cheung, M. Anikin, P. Cramer, and D.

Temiakov, A novel intermediate in transcription initiation by human

mitochondrial RNA polymerase, Nucleic Acids Research. 2014; 42 (6), 3884-

3893. doi:10.1093/nar/gkt1356.