1

Ribosome frameshifting and stalling stimulated

by 22 base pair mRNA stem-loop structures

Sigrid Stokbro Hess

June 2013

Supervisor:

Michael Askvad Sørensen

Department of Biology

Picture ref: http://cronodon.com/BioTech/Ribosomes.html

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Indholdsfortegnelse 1. Preface ....................................................................................................................................................... 3

2. Abstract ..................................................................................................................................................... 4

3. Resumé ...................................................................................................................................................... 5

4. Introduction ............................................................................................................................................... 6

4.1. Translation ......................................................................................................................................... 6

4.2. -1 programmed frameshift: ............................................................................................................... 6

4.3. Models for programmed -1 frameshift ............................................................................................. 7

4.4. Pseudoknots as frameshifting signals ................................................................................................ 8

4.5. Stem-loop structures as frameshift signals ....................................................................................... 9

4.6. Models for the difference between stem-loops and pseudoknots ................................................. 10

4.7. The results of Tholstrup et al. (2012) .............................................................................................. 11

4.8. My study .......................................................................................................................................... 11

5. Materials and methods ........................................................................................................................... 11

5.1. Strains .............................................................................................................................................. 11

5.2. Making of competent cells .............................................................................................................. 12

5.3. Cloning of the DNA constructs ........................................................................................................ 12

5.4. Construction of plasmid .................................................................................................................. 13

5.5. Examining the sequence of pSSH1 and pSSH2 ................................................................................ 14

5.6. Frame-shift assay ............................................................................................................................. 15

5.7. One dimensional gels ...................................................................................................................... 16

5.8. Nonequilibrium two dimensional gels ............................................................................................. 16

6. Results ..................................................................................................................................................... 17

6.1. Plasmids ........................................................................................................................................... 17

6.2. Frameshifting ................................................................................................................................... 19

6.3. Stalling of the ribosomes ................................................................................................................. 20

7. Discussion ................................................................................................................................................ 22

7.1. Modeling the inserted constructs ................................................................................................... 22

7.2. The inserted constructs were able to stimulate frameshift ............................................................ 24

7.3. The inserted constructs were able to stimulate ribosomal stalling ................................................ 26

8. Conclusion ............................................................................................................................................... 26

9. Future studies: ......................................................................................................................................... 27

10. References ........................................................................................................................................... 28

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1. Preface This Bachelor study was conducted in the period from February 2013 to June 2013 at the Department of

Biology, University of Copenhagen, under the guidance of Ass. Professor Michael Askvad Sørensen.

I truly appreciate all the help he provided during the entire process. In addition, I would like to thank Marit

Warrer, laboratory technician, for tutoring me in laboratory procedures and helping me with various

experiments and Mette Konstad for her constructive criticism during the process of writing this thesis.

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2. Abstract It has long been established that programmed -1 frameshift plays an important physiological role for some

viruses and bacteria. -1 frameshift occurs when the ribosome moves 2 instead of 3 bases during a

translational elongation cycle. A frameshift signal placed between two slightly overlapping genes (the

second gene in the -1 reading frame with respect to the reading frame of the first gene) ensures a fixed

ratio of the two protein products, making programmed frameshift an important regulatory mechanism. A

frameshift signal consists of a slippery sequence of 7 nucleotides, a spacer of 6 to 9 nucleotides and an RNA

structure preventing a fast ribosomal read-through. The pseudoknot is a well known frameshift signal, but

in some genes like the Escherichia Coli dnaX, the frameshift signal is believed to be a stem-loop structure.

Despite of extensive research of programmed -1 frameshift, the mechanism behind it is still largely

unknown. Recent findings reveal that in addition to promoting frameshift, pseudoknots are also able to

stimulate ribosomal stalling. Stalling occurs when a frameshifted ribosome is unable to unwind the

pseudoknot and is paused at the frameshift signal indefinitely. In the present study, I examine whether

expected stem-loop structures induce frameshift and stalling of the ribosome. The sequences, predicted to

form stem-loop structures, were derived from a sequence predicted to form a pseudoknot structure known

to stimulate both frameshift and ribosomal stalling. By radioactive labeling the proteins from bacteria,

transformed with a reporter plasmid containing the predicted stem-loops, I obtained the frameshift

efficiency of the analyzed structures and assessed their stalling ability. The analyzed constructs proved to

be able to induce both ribosomal frameshifting and stalling, but theoretical structural modeling of the

analyzed constructs, casted doubt about the actual structure of the predicted stem-loops. As the actual

structure of the examined constructs is debatable it was not possible to draw a conclusion as to whether a

stem-loop structure is sufficient to induce ribosomal frameshift and stalling.

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3. Resumé Det er et velkendt fænomen at programmeret -1 frameshift spiller en vigtig fysiologisk rolle for nogle vira

og bakterier. -1 frameshift forekommer når ribosomet rykker 2 i stedet for 3 baser i løbet af en

elongeringscyklus. Et frameshiftsignal placeret mellem to let overlappende gener (det andet gen i -1

læseramme i forhold til det første gens læseramme) sikrer en fast ratio af de to proteinprodukter. Denne

funktion gør programmeret frameshifting til en vigtig regulativ mekanisme. Et frameshiftsignal består af en

slippery(glat) sekvens, en spacer og en RNA struktur der forhindrer ribosomet i at læse hurtigt igennem

signalet. Pseudoknuden er et velkendt frameshiftsignal, men i nogle gener, som f.eks. Escherichia Coli dnaX,

mener man at frameshiftsignalet er en stem-loopstruktur. På trods af omfattende forskning på området er

mekanismen bag programmeret -1 frameshift stadig stort set ukendt. For nyligt blev det opdaget at, udover

at fremkalde frameshift, kan pseudoknuder også stimulere stalling af ribosomer. Stalling opstår når det er

umuligt for et frameshiftet ribosom at læse igennem pseudoknuden og det derfor bliver standset ved

frameshiftsignalet på ubestemt tid. I dette studie undersøger jeg om forventede stem-loop strukturer

stimulerer frameshift og stalling af ribosomet. Sekvenserne, der forventes at danne stem-loop strukturer,

er afledt af en sekvens forudsagt til at folde som en pseudoknude, der tidligere har stimuleret frameshift og

ribosomal stalling. Ved at radioaktivt mærke proteiner fra bakterier transformeret med plasmider

indeholdende de forventede stem-loop strukturer, fandt jeg frameshifteffektiviteten for de forventede

stem-loop strukturer og vurderede deres evne til at inducere ribosomal stalling. De analyserede strukturer

viste sig at være i stand til både at fremkalde ribosomal frameshift og stalling, men teoretiske strukturelle

modelleringer af strukturerne såede tvivl omkring den reelle struktur af de forventede stem-loop

strukturer. Da det kan diskuteres hvilken struktur de analyserede konstrukter reelt har, var det ikke muligt

at drage en konklusion om hvorvidt en stem-loop struktur kan stimulere ribosomal frameshifting og stalling.

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4. Introduction Despite extensive research in the field of programmed -1 frameshift, the precise mechanism behind this

phenomenon is still largely unknown. In order to understand the different models and hypotheses

regarding -1 programmed frameshift, it is important to understand the basics, being the mechanism of

translation.

4.1. Translation The bacterial ribosome consists of a large and a small subunit. Combined they create a tunnel which the

mRNA travels through and three sites that contain the tRNA: the A-site, the P-site and the E-site. At the

beginning of the elongation cycle an aa-tRNA, in a complex with Elongation Factor Tu (EF-Tu) and GTP,

enters the ribosome at the A-site. The P-site already contains the tRNA linked to the amino acid last added

to the growing peptide chain. If the tRNA anticodon matches the mRNA codon, a conformational change of

ribosome stabilizes the binding of tRNA and initiates GTP-hydrolysis by EF-Tu, which leads to the release of

the aminoacyl end of the A-site tRNA. Next step is called accommodation in which the A-site tRNA distorts

so its aminoacyl end swings into the P-site. In the ribosomal peptidyl transferase site, a peptide bond is

formed between the growing peptide chain and the aa-tRNA from the a-site. After the formation of the

peptide bond the P-site tRNA is deacylated. Next step is translocation in which the mRNA moves exactly

one codon(3 bases) so the A-site tRNA (bound to the peptide chain) moves to the P-site, and the deacylated

P-site tRNA moves to the E-site. Hydrolysis of GTP by Elongations Factor G (EF-G) drives translocation. The

translocation of the small and big subunit takes place separately which leads to hybrid states of tRNA

position in the ribosome. For example the A/P state refers to a tRNA position when the big subunit has

translocated, but the small subunit has not. This leaves the tRNA in the A-site of the small subunit but in the

P-site of the big subunit. The elongation follows the initiation and is followed by termination, but neither is

essential to the understanding of frameshifting and will not be described in this paper. (Reviewed by

Ramakrishnan, 2002, Giedroc et al. 2009, Schmeing et al. 2009)

4.2. -1 programmed frameshift: Frameshift occurs when the ribosome, after completed elongation cycle, fails to move exactly one codon

either caused by a mistake or by programmed frameshift (reviewed by Farabaugh 1996). In most cases

frameshift leads to a production of flawed proteins, but in some cases, it is an important regulatory

mechanism. This was discovered in a study of the bacteriophage MS2 in 1982(Kastelein et al. 1982). For

programmed frameshifting to take place it requires a signal. Such a signal consists of 3 motifs. A slippery

sequence and a spacer (6-9 nucleotides) located directly upstream from a RNA structure hindering the

ribosome from reading fast trough it. The slippery sequence consists of 7 nucleotides of the form XXXYYYZ,

as seen in the coronavirus Infectious Bronchitis Virus (IBV) UUUAAAC (Brierley et al. 1989). The RNA

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structure troubling the ribosome could be a pseudoknot, a hairpin/stem-loop structure or something else

entirely (Reviewed by Farabaugh 1996, Giedroc et al, 2009).

4.3. Models for programmed -1 frameshift The mechanism behind programmed -1 frameshift is poorly understood. The following models each have

an idea as to how the frameshift process functions.

The passive model: In the passive model, frameshift is induced by simply pausing above the slippery

sequence. Some correlation between pausing of the ribosome and frameshift efficiency has been found

(Somogyi et al. 1993), but this is not always the case and more than simply pausing over the slippery

sequence seems to be necessary for frameshift to occur(Kontos et al. 2001).

The hassled EF-G model: This model proposes that the frameshift signal hinders EF-G from fully entering

the ribosome. By blocking the entrance into the mRNA tunnel, the pseudoknot reduces the effect of EF-G,

leading to a translocation of two, not three, bases (Weiss et al. 1989, Yusupova et al. 2001). The advantage

of this model is that the energy needed to frameshift is well accounted for, however mutating the

eukaryotic equivalent to EF-G does not affect frameshift ( Hudak et al. 2001).

The mechanical model: Mechanical models consider mRNA tension to be the triggering factor. The

following two models are mechanical models.

The 9 Å solution suggests how the possible mRNA tension arises. When the aa-tRNA moves from the T/A

state to the A/A state it moves 9 Å. As it is connected to the mRNA, the mRNA string must move these 9Å

as well. If the pseudoknot is not unwound, a tension in the mRNA string emerges. This tension can be

relieved by a -1 frameshift. This places the frameshift before peptidyl transfer but after or during intake of

aa-tRNA (Plant et al. 2003).

A study of ribosomes paused at a pseudoknot agrees that the tension of mRNA is vital to the process of

programmed frameshift, but places the time of frameshift during translocation. The movement of tRNA

from the A site to the P site during translocation is hassled by the mRNA tension. It is not possible for the

tRNA to return to the A site as this is preoccupied by the eEF2(eukaryotic EF-G). The opposing forces of the

mRNA ‘pulling’ and the ribosome ‘pushing’ the tRNA, lead to a bent conformation of the tRNA. This leads to

a break of the codon-anticodon interaction and the restoring of the normal tRNA conformation leads to a

new codon-anticodon interaction in the -1 reading frame (Namy et al. 2006).

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4.4. Pseudoknots as frameshifting signals The pseudoknot was discovered in the 1980’s (Pleij et al 1985, Rietveld et al. 1983) and the 3-dimentional

structure was characterized by NMR in 1998 (kolk et al. 1998). The importance and dispensability of the

different features of the pseudoknot have been investigated by many. The secondary structure of the

pseudoknot is believed to be essential for the frameshift efficiency as changing primary structure in the

pseudoknot was not found to affect the frameshift efficiency (Brierley et al. 1991). The most studied

pseudoknot, the H-type pseudoknot is formed when nucleotides within a hairpin loop base pair with

nucleotides 3’ from the hairpin, and consist of a stem 1, loop 1, loop2 and a stem 2 (fig.1).

Figure 1: WT IBV pseudoknot belonging to the large class of pseudonots (Naptine et al. 1999).

The H-type pseudoknot can be subdivided into two major classes: A larger class characterized by a long

stem 1 and a smaller class characterized by a short stem 1 and extensive loop2 stem 1 interactions (Chou

and Chang 2010; Giedroc and Cornish 2009)

The large class of H-type pseudoknots: For the large class pseudoknot, a minimum length of stem 1 seems

to be important to stimulate frameshift. Decreasing the stem 1 length from 11 to 10 base pairs dramatically

lowers the frameshift efficiency even though it does not abolish the pseudoknot structure (Napthine et al.

1999). Stem 2 seems important as well since destabilizing stem 2 leads to a decrease of frameshift

efficiency ( Brierley et al. 1991). Pseudoknots of the large class are believed to have an overall structure

with stem 2 stacked on top of stem 1(Michiels et al. 2001).

The small class of H-type pseudoknots: in contrast to the large class of pseudoknots, stem 1 length is not

as important for the small class of pseudoknots. The overall structure of the pseudoknot however, seems of

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great importance for frameshift efficiency. A single Adenine residue in the junction between the two stems,

contributing to an overall bent structure of the pseudoknot, seems to be of importance to frameshift

efficiency for these pseudoknots (Kang et al. 1997, Chen et al.1996). Adding this A residue in the junction

between the stems along with an A residue at the 3’-end of loop 2 (loop 2, stem 1 interaction) makes it

possible to bypass the requirement of an 11 bp stem 1 in the large class of pseudoknots. The need for the A

residue at the end of loop 2 is not present if loop 2 is 14 nucleotides long (Liphardt et al. 1999) perhaps

because a long loop 2 allows for even more loop 2 stem 1 interaction.

The strength of pseudoknots: When wanting to measure the strength of a frameshift signal, one

encounters the problem of whether to take the energetically or mechanically approach. In the energetically

approach the strength is measured solely as thermodynamic stability and while studies have shown

correlation between the thermodynamic stability and the frameshift efficiency in some of the cases (Ten

Dam et al. 1995, Bidou et al. 1997) this correlation is not always found (Cao et al. 2008). The mechanical

approach includes both the thermodynamic stability and the theoretical work needed to stretch the RNA

structure when measuring the strength of a pseudoknot. Hansen et al. (2007) uses optical tweezers to pull

the pseudoknots apart and find a correlation between mechanical force required to unwind the

pseudoknot and frameshift efficiency. The work needed in practice to unfold the pseudoknots exceeds the

calculated theoretical work needed, explaining the different frameshift efficiency between the previously

mentioned (see section on large class pseudoknots) pseudoknots with 11 or 10 base pairs in stem 1. This

finding indicates that more than thermodynamic stability and the ‘theoretical stretching work’ is needed to

explain the strength of a pseudoknot.

4.5. Stem-loop structures as frameshift signals It is still uncertain whether a stem-loop structure is sufficient to stimulate frameshift or not, nonetheless

frameshift signals in the form of stem-loops have been found.

Stem-loop frameshift signals in viruses and bacteria: The frameshift signal in E.coli dnaX is believed to be a

stem-loop structure (Tsuchihashi et al. 1991, Larsen et al. 1997) and in some viruses, for example HIV-1,

CfMV and SIV, the stem-loop structure has shown to be sufficient to promote frameshift (Gaudin et al.

2005, Lucchesi et al. 2000). However other studies of frameshift signals from HIV-1 and BYDV contradicts

the previous assumption that those signals are simple stem-loop structures (Barry et al. 2002, Dinman et al.

2002, Dulude et al. 2002). Supporting the believe that a stem-loop functions as a frameshift signal, Larsen

et al. (1997) found a correlation between theoretical strength and frameshift efficiency of different

mutated versions of the dnaX stem-loop structure.

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If the stem 1 length is the most important feature for the large class pseudoknots (Napthine et al. 1999),

the stem-loop structure might be sufficient as a frameshift signal, as stem 1 in a pseudoknot basically is a

stem-loop. Brierley et al. (1992) suggest that a stem-loop structure might be sufficient to promote

frameshift perhaps aided by a slippery sequence of an even more slippery character: UUUUUUA.

If programmed -1 frameshift occurs as proposed in ‘the hassled EF-G model’ (see section on models for

programmed -1 frameshift), one might imagine that a stem-loop structure could match a pseudoknot when

it comes to physically blocking the EF-G from entering the ribosome. However, as seen in the next section,

other models propose that the different characteristics of the two structures affect the mechanism of

frameshifting.

4.6. Models for the difference between stem-loops and pseudoknots The apparent difference between a stem-loop and a pseudoknot is the forming of stem 2, but other

differences like loop2-stem1 interactions might be present as well. If and how these differences is

important is not known for certain. The following is suggestions as to how these differences might manifest

themselves in the context of programmed -1 frameshift.

Torsional restraint: A model by Dinman et al. (1995) focuses on torsional restraint. The model proposes

that unwinding the stem 1 requires rotation, and that the freedom to rotate presumably is restricted by the

nucleotides in loop 1 taking part in the stem 2 base pairs. Because of this restricted freedom to rotate, the

ribosome would have to pause to unwind the pseudoknot and would be positioned over the slippery

sequence (Dinman 1995). The observation that decreased rotational freedom leads to higher frameshift

efficiency supports this model (Plant et al. 2005).

Bent conformation of tRNA: When developing their frameshift model, Namy et al. (2006) found that a

ribosome paused by a stem-loop structure does not result in the bent conformation of the P-site tRNA they

see in ribosomes paused by a pseudoknot (the Mechanical model). They propose that a stem-loop is less

able to promote frameshift because the ribosome is able to unwind it 3 bases at a time (in pace with

translocation) and as a result, the mRNA tension that leads to the bent conformation of the tRNA is absent.

Though the findings of naturally occurring stem-loop frameshift signals (see the section on stem-loops as

frameshift signals) disagrees with these models, the finding that a stem-loop structure results in lower

frameshift efficiency compared to a pseudoknot (Somogyi et al. 1993) supports that a functional difference

between stem-loops and pseudoknots affects the process of programmed -1 frameshift.

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4.7. The results of Tholstrup et al. (2012) My study is based on the research of stalling ribosomes by Tholstrup et al. (2012). Stalling of the ribosome

occurs if the frameshifted ribosome is unable to unwind the pseudoknot and the pseudoknot-induced

pausing (seen in Somogyi et al. 1993 and Kontos et al. 2001) continues indefinitely.

Tholstrup et al. transforms MAS90 with a reporter plasmid containing gene 10, a pseudoknot structure (of

various kind) and -1 reading frame lac-Z. By conducting a pulse-chase assay and separating the proteins by

nonequilibrium 2 dimensional SDS gel they investigates stalling of ribosomes.

The study shows that all the included pseudoknots are able to promote frameshift. The results further

demonstrate that the pseudoknots stall ribosomes and that enhanced strength of the pseudoknot base

pairs increases the ability to stall ribosomes. Of the investigated pseudoknots, the pseudoknot that

generates most stalling is 22/6a (fig. 3).

Tholstrup et al. (2012) propose that neglecting to take stalling of the ribosome into account, might lead to

underestimating of the frameshift efficiency, as it is impossible to distinguish the non-frameshifted product

from the frameshifted but stalled product. Considering stalling when calculating the 22/6a frameshift

efficiency more than doubles the result, so especially when dealing with strong pseudoknots, stalling is an

important factor to include (Tholstrup et al. 2012).

4.8. My study While some studies have found different stem-loop structures to be functional frameshift signals, others

cast doubt on whether those structures in fact are simple stem-loop structures. The purpose of this study is

to address the question of whether a simple stem-loop structure, based on the structure of stem1 in the

22/6a pseudoknot from the research by Tholstrup et al. (2012), stimulates -1 frameshift and/ or stalling of

the ribosome.

5. Materials and methods

5.1. Strains NF1830: laboratory strain: MC1000 (Casadaban et al. 1980)made recAI, F´ lacIqI, lacZ::Tn5, proAB+

MAS90: recAI, Δ(lac, proAB), thi-, rel+, F´ lacIqI, lacZ::Tn5, lacYA+, proAB+(Tholstrup et al. 2012, Hansen et al.

2007)

JT543: NF1830 + pJT516(OFX_302, containing slippery sequence, spacer and pseudoknot 22/6b)(Tholstrup

et al. 2012)

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JT538: MAS90 +pJT515(OFX_302, containing slippery sequence, spacer and pseudoknot 22/6a)(Tholstrup et

al. 2012)

T439: MAS90 + pTH421(pOFX_302, containing slippery sequence, spacer and 4 nucleotides)(Hansen et al.

2007, Tholstrup et al. 2012)

SSH3: MAS90 + pSSH1(pOFX_302, containing slippery sequence, spacer and stemloop 22a)

SSH4: MAS90 + pSSH2(pOFX_302, containing slippery sequence, spacer and stemloop 22a/rev6revUA)

5.2. Making of competent cells 5 mL over night culture of NF1830 was diluted into 500 mL YT media and incubated with shaking at 37oC.

OD was measured with a spectrophotometer regularly and plotted on a semi log paper to ensure the

growth was exponential and to estimate the time the OD436 is between 0,6 and 0,8. When the OD436 was

approx. 0,7 the culture was put on ice for 20 minutes and swirled to make sure all of the culture was cooled

down. The culture was centrifuged at 3.000 G for 15 minutes, the supernatant was disposed of and the

pellet was resuspended in 200 mL 100 mM cold CaCl2. The cells were kept on ice for 20 minutes and then

centrifuged at 3.000 G for 10 minutes. The supernatant disposed of and pellet was resuspended in 45 mL

100mM cold CaCl2. The cells were left at 25oC for 20 minutes, centrifuged at 3.000 G for 10 minutes and

resuspended in 100 mL 100mM CaCl2. 1,2 mL 50 % glycerol was added and the cells were divided into

portions of 200 μL and froze by N2 in Eppendorf tubes and store in -80oC.

Competent cells were sometimes made in small scale and used immediately.

5.3. Cloning of the DNA constructs The ordered DNA sequences (22a and 22a/rev6revU) were dissolved in 50 μL TE-buffer. 1 μL of the DNA

solution was added to 100 μL competent NF1830 cells and left on ice for 20min.The cells were plated on

100γ Ampicillin YT agar plates and competent cells without added DNA were plated as a control. JT543, a

strain containing the reporter plasmid needed in the frame shift assay, was streaked on a 100γ Amp YT agar

plate. The agar plates were left for incubation at 37oC over night.

Next day the strains were once again streaked on 100γ Amp YT agar plates and left to incubate at 37oC over

night, to make sure we proceeded working with the correct cells. One colony of each kind was grown into a

fluid culture in 5mL 100γ Amp YT media and incubated with shaking at 37oC over night.

Using ENZA plasmid mini kit I D6942-02 the plasmids were isolated from the fluid cultures and stored at

approx. 4o C.

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5.4. Construction of plasmid

To examine if the restriction enzymes worked, the plasmids were cut with either HindIII or ApaI and

separated by electrophoreses on a 0,7 % agarose gel along with the uncut plasmids. The three

plasmids(plasmid containing 22a, plasmid containing 22a/rev6revU and pJT516) were cut with both HindIII

and ApaI by mixing either 8 μL 22a, 8μL 22a/rev6revU or 4 μL pJT516 with 1μL buffer 4, 1μL ApaI and ½ μL

HindIII. The mix was left for an hour at 25oC and an hour at 37oC. Alkaline phosphate was added to pJT516.

To isolate the small fragments (22a and 22arev6revU) the mix was run on a 2% agarose gel at voltage 110.

To isolate the reporter plasmid (pTJ516 without the pseudoknot) the mix was run on a 0,7 % agarose gel at

voltage 110. The bands were visualized with UV light for as short a time as possible, and cut out of the gel

with a scalpel.

Electro elution: The pieces of gel were placed in dialysis sacks filled with ½xPB buffer. The dialysis sacks

were closed with clamps and the DNA was extracted from the pieces of gel by electrophoresis. The PB

buffer from the dialysis sacks (approx. 300μL), now containing the DNA fragment, were transferred to

Eppendorf tubes and 150μL phenol was added before vortexing and centrifugation at 20.000G for 1½

minute. The upper liquid layer, containing the DNA, was collected and transferred to a new tube. 300μL

chloroform was added to each tube. After shaking the tubes a new upper layer formed which was

transferred to new Eppendorf tubes. 20μL 5 M NaCl was added to each tube ensuring that the

concentration of the final volume was 100 mM. Finally 700μL Ethanol was added to the mix.

Ligation: The precipitated DNA in ethanol was centrifuged at 20.000 G for 15 minutes and the supernatant

was removed. 200 μL ethanol was poured down the sides of the tube with the pellet and disposed of. The

tubes with pellets were dried at 65o. pJT516 was dissolved in 10 μL 1x ligation buffer (10 x ligation buffer:

600mM Tris∙HCl pH 7,5 and 100mM MgCl) and 22a and 22a/rev6revU was dissolved in 5 μL 1x ligation

buffer each. To promote ligation of 22a or 22a/rev6revU into the reporter plasmid a ligation mix was

prepared by mixing 2,5 μL ligation buffer, 2,5 μL 20 mM ATP, 5 μL 100 mM DTT, 15 μL 2% gelatin and 1 μL

ligase. To make pSSH1, 5μL of 22a was mixed with 3 μL pJT516 and 8 μL ligation mix. To make pSSH2, a

similar portion was made with 22a/rev6revU and a control was mixed from 3 μL pJT516, 5 μL 1x ligation

buffer and 8 μL ligation mix. To control if the ligation mix worked, 1 μL λBstEII was added to 5 μL ligation

mix with ligase and 1 μL λBstEII was added to 5 μL ligation mix without ligase. The tubes were incubated

over night at 16oC.

8 μL of both pSSH1 and pSSH2 was mixed with 100 μL competent cells. They were left on ice for 20

minutes, heated in a heating block at 42oC for 90 seconds and plated on100γ Amp YT agar plates. The

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plated were incubated at 37oC over night. The following day SSH1 (a NF1830 colony containing pSSH1) and

SSH2 (a NF1830 colony containing pSSH2) were streaked on 100γ Amp YT agar plates and incubated at 37oC

over night. The two strains were grown as a liquid culture in 100γ Amp YT media and incubated with

shaking at 37oC over night. Using ENZA plasmid mini kit I D6942-02 the plasmids were isolated from the

fluid cultures and stored at approx. 4o C.

5.5. Examining the sequence of pSSH1 and pSSH2 Using the web based software Diffcutter (see references) the restrictions enzymes that digests pSSH1 and

pSSH2 differently were found as well as restriction enzymes that differs between pOFX_302 (pJT516 ligated

back together without the inserted pseudoknot) and pSSH1 or pSSH2. pSSH1 and pSSH2 were digested with

BssHII and pSSH1, pSSH2 and pOFX_302 were digested with PvuII. The BssHII digests were separated on a

0,7 % agarose gel at voltage 110 and the PvuII digests were sererated on a 2 % agarose gel at voltage 110.

The number and positions of bands were as expected. To further confirm the plasmids sequences they

were sent to Eurofins for sequencing. The concentrations of DNA in the plasmid solutions were measured

at Thermo Scientific NanoDrop 1000 spectrophotometer and were approx. 50 ng/μL. The plasmids were

mixed with primers that amplify the inserted construct; TH412 and OFX302_1. 17 μL plasmid (pSSH1 or

pSSH2) were mixed with o,5 μL primer(TH412 or OFX302_1) in an Eppendorf tube with a prepaid label and

sent to Eurofins lab. Enzymes used were bought from Fermentas and New England Biolabs and used as

recommended by the manufacturer.

Figure 2: A: lane 1: marker, lane 2: pOFX_302(8062), lane 3 and 4: pSSH1(6571, 1508), lane 5 and 6: pSSH2(6571, 1508). Plasmids were cut with BssHII. B: lane 1:marker, lane 2 and 3: pSSH1(4267, 2557, 950, 363), lane 4 and 5: pSSH2(4168, 2557, 950, 363, 138). Plasmids were cut with PvuII.

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5.6. Frameshift assay The frame-shift assays were conducted in MAS90 cells. A culture of MAS90 was made competent and 100

μL of cells was mixed with 8 μL pSSH1 or pSSH2 and was plated on 100γ Amp YT agar plates. As a control 50

μL MAS90 without added plasmid was plated in a corner of one of the agar plates. The plates were

incubated at 37oC over night. The following day the successfully transformed colonies SSH3 and SSH4 as

well as T439 and JT538 were streaked on 100γ Amp YT agar plates. The plates were incubated at 37oC over

night.

The 4 strains were grown to a liquid culture in MOPS media (1xMOPS, 100γ Ampicillin, 15γ Kanamycin, 0,4

% glycerol, 5γ B1 +K2PO4). The cultures were incubated with shaking at 37oC over night. The cultures were

grown exponential for around 24 hours before frame-shift assay. 20 μL of overnight culture was diluted in 2

mL preheated MOPS media. They were grown with shaking at 37oC for 3 hours after which 500 μL were

diluted in 5 mL preheated MOPS media. The cultures were incubated with shaking at 37oC for 4 hours. OD

was measured and the dilution necessary to reach OD436 0,5 at the start of frame-shift assay was calculated.

The cultures were incubated with shaking at 37oC until start of frame-shift assay.

The cultures OD was measured and they were transferred to preheated 100 mL flasks positioned in a

shaking 37oC water bath. The cultures OD436 was measured approx. every 20 minutes and plotted on a semi

log paper to ensure the growth was exponential. 5 mL culture was transferred to a new preheated 100 mL

flask in the water bath and induced with 50 μL 0,1 M IPTG for 15 minutes. Approx. 10 minutes after the

induction, 1 mL culture was transferred to a preheated plastic vial also positioned in the water bath.

Precisely 15 minutes after the induction 0,5 μL radioactive S35Methionine was added to the vial (the pulse).

10 seconds later 10 μL cold Methionine was added to the vial (the chase). 2 minutes after adding the cold

Methionine, the labeled culture was transferred to an Eppendorf tube on ice with 15 μL cold 100 mg/mL

chloramphenicol. JT538 was labeled before and after induction with IPTG but SSH3, SSH4 and T439 was

only labeled after induction with IPTG.

The labeled cultures were centrifuged at 20.000 G for 2 minutes and the supernatants were disposed of. 1

mL sodium dodecyl sulfate(SDS) loading buffer (1:1:1 10 % SDS, 4x stacking buffer(see section on one

dimensional gel), 30 % sucrose and 5 μL DDT/ml) was mixed with 0,5 μL 1M DDT. The pellets were

resuspended in 40 μL of boiling buffer with DDT. The labeled proteins were stored at approx. -20oC.

One frame-shift assay was conducted with a 10 second pulse and one with a 20 second pulse.

16

5.7. One dimensional gels The proteins were separated on a one dimensional SDS polyacrylamide gel electrophoresis (PAGE). The

8,75 % separation gel was made from 11,7 mL 30 % acrylamid/0,8 % bisacrylamid, 10 mL separation buffer

pH=8,8 (182g trizma base, 25 mL HCl, 1 L Millipore water), 0,4 mL 10 % SDS, 0,4 mL 10 % APS and 17,3

Millipore water. A mould was made from two glass plates, two spacers and a plastic sheet. 5 μL TEMED was

added to 5 mL separation mix and this was poured down the side of the spacers to seal the mould. 20 μL

TEMED was added to the rest of the separaion mix and this was poured into the mould. Approx. 1 mL water

was poured very slowly on top of the gel. This was left to polymerize and 5 % stacking gel was made from

1,7 mL 30 % acrylamid/0,8 % bisacrylamid, 2,5 mL stacking buffer pH=6,8 (61g trizma base, 19 mL HCl, 1 L

Millipore water(18.2 MΩ•cm resistivity at 25 °)), 0,1 mL 10 % SDS, 50 μL 10 % APS and 5,7 mL Millipore

water. This was mixed with 5 μL TEMED, the water was removed from the separation gel and the stacking

gel mix was poured on top of it and the wells were casted.

2 μL 0,01 M DDT was added to the 40 μL labeled proteins and boiling buffer. They were boiled at 100oC for

2 minutes in a heating block. The gel was placed in 1x EL buffer (10xEL buffer; 30,2g trizma base, 144g

glycin, 1L Millipore water) and 12 μL of each sample were loaded into the wells. The applied voltage started

at 120 V but was gradually increased to 450V. When the blue color (from the boiling buffer) reached the

bottom of the gel, the electricity was turned off and the gel was placed in HCl until the blue color changed

to yellow. The gel was transferred to water and when the color was blue again the gel was transferred to

3mm Wathman paper and dried by heat and vacuum. The paper with the gel was placed in a Molecular

Dynamics phosphor screen and left for 1-3 days. The phosphor screen was scanned at a Molecular Dymaics

840 Storm scanner.

The frameshift efficiency was calculated as follows:

5.8. Nonequilibrium two dimensional gels To investigate whether stalling had taken place the proteins were separated on a two dimensional SDS

PAGE. O´Forrell gels to separate the proteins according to charge was made from 2,75g ultra pure UREA,

0,66 mL 30 % acrylamid/0,8 % bisacrylamid, 1 mL 10 % NP40, 900 mL Millipore water and 300 μL

ampholines pH 3,5-10. 15 μL 10 % APS and 12 μ TEMED were added and the mix was sucked into small

pipes (approx. 400 μL in each pipe) and left to polymerize. The pipes were placed with bottom end in 20

mM NaOH and the top end in 10 mM H3PO4. 15 μL 10 % NP40 and 1 μL 30 mg/mL RIM was added to each

17

sample and the samples were saturated with urea. 15 μL of sample was loaded into each pipe. The gels

were left running for 1200 Volt hours. The O´Forrell gels were blown out of the pipes and into sample

buffer (4,6g pure SDS, 24 mL 87% glycerol, 25 mL stacking buffer, 10 mL mercaptoethanol,

bromphenolblue, millipore water to total volume of 200 mL).

8,75 % SDS gels were made, run and scanned as described in the section on one dimensional gels, except

the stacking gel was replaced by the O´Ferrell gels which was placed on top of the separation gel.

6. Results

6.1. Plasmids To study whether a simple stem-loop is sufficient to promote frameshift and/or stalling of the ribosome,

two versions of stem1 in pseudoknot 22/6a (Tholstrup et al. 2012) were created. 22a (fig. 3) stops

immediately after the stem-loop and 22a/rev6revU (fig. 4) continues after the stem-loop but the 6 base

pairs, that create stem2, and 6 other bases, that creates an alternative stem2, are disrupted. The constructs

have an in-frame stop codon placed in the spacer between the slippery sequence and the stem-loop (fig. 3

and fig. 4).

Figure 3: Expedted structure of Pseudoknot 22/6a (Tholstrup et al. 2012) and 22a. Red: slippery sequence. Blue: spacer.

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Figure 4: Expected structure of 22a/rev6revU and of the structure inserted in pTH421. Red: Slippery sequence. Blue: spacer.

The two sequences predicted to form stem-loop structures were placed in a reporter plasmid containing an

IPTG inducible promoter, T7 gene10, the inserted element and lacZ (in -1 reading frame with respect to the

reading frame of gene10) (fig.5). The plasmid also contains the selective marker bla making the cells

transformed with the plasmid resistent to ampicillin. The in-frame Stop product produced from this plasmid

is a 28kDa polypeptide and the product produced in case of frameshift is a 148kDa polypeptide fusion

between the product of gene10 and β-Galactosidase (fig. 6 and fig. 8).

Figure 5: Reporter plasmid with IPTG inducible promoter, T7 gene10, the inserted element, lacZ, ColE1 origen of replication and bla.

19

6.2. Frameshifting The promoter in the reporter plasmid was induced with IPTG and the proteins labeled with radioactive L-

S35methionine. The radioactive labeled proteins from the strains JT538, SSH3, SSH4 and T439 were

separated on a one dimensional SDS gel to examine the frameshift efficiency (fig. 6) As expected the

frameshift product was present in JT538(lane2), but not in T439(lane5). The band representing the

frameshift product was also present in lane 3, containing proteins from SSH3, and in lane 4, containing

proteins from SSH4, indicating that the inserted constructs were able to stimulate ribosomal frameshift.

The calculated average frameshift efficiency was 1.5 % (±0.8) for the 22/6a pseudoknot, 1.4 %(± 0.7) for

22a, 1.4 % (±0.3) for 22a/rev6revU and 0.6 % (±0.3)for the control consisting of 4 nucleotides. Only two

experiments have been carried out, hence the high standard deviation. The first experiment led to a

frameshift efficiency of approx. 2 % and the second experiment led to a frameshift efficiency of approx. 1

%. The only deliberate difference between experiment t one and two was the pulse time, which was 10 and

20 seconds respectively.

Figure 6: A: One dimensional gel: lane1: JT538 - IPTG, lane 2: JT538 + IPTG, lane 3: SSH3 + IPTG, lane 4: SSH4 + IPTG, lane 5: T439 + IPTG. B: Graph displaying frameshift efficiency.

20

I found that the frameshift efficiency of the predicted stem-loop structures was practically the same as the

frameshift efficiency of the 22/6a pseudoknot (fig. 3), pointing to the conclusion that in this case the

pseudoknot was not vital to the frameshift process.

6.3. Stalling of the ribosomes To study whether the two constructs were able to stall the ribosome, the labeled proteins from the

different strains were separated on a two dimensional SDS gel. The two dimensional gel separates the

proteins according to pI and weight (fig.7 and fig. 8). At first I separated the proteins on an equilibrium two

dimensional gel, but the stalled product were too difficult to distinguish from other proteins. This issue was

resolved by separating the proteins on a nonequilibrium two dimensional gel. A difference was found

between the nonequilibrium two dimensional gels of proteins from the control strains (JT538 and T439)

(fig. 9). Spots representing proteins at about the same size as the in-frame stop product but with a higher pI

were seen at the JT538 gel. Those spots were not present at the T439 gel. The proteins corresponded to the

expected size and pI of growing frameshift products with stalled ribosomes (Tholstrup et al. 2012). As seen

from fig. 10, the spots representing the stalled products were present on the gels with proteins from SSH3

and SSH4.

Figure 7: Two dimensional gel with proteins from JT538 before adding IPTG. OmpF:37kDa, Tu:43kDa, GroEL:57kDa, DnaK:69kDa and EfG:78kDa. Protein size was found at internet based databases (http://www.ecogene.org/ and http://www.uniprot.org/).

21

Figure 8: Nonequilibrium two dimensional gel. JT538 + IPTG.

Figure 9: Nonequilibrium two dimensional gels. JT538 - IPTG, JT538 + IPTG and T439 + IPTG. Arrows pointing out the area with stalled products from JT538 and the area they could have been at the gel with proteins from T439.

22

Figure 10: Nonequilibrium two dimensional gels. SSH3 + IPTG, SSH4 + IPTG. Arrows pointing out the area with stalled products.

In summery my results from the frameshift assay and the stalling assay indicate that 22a and 22a/rev6revU

were able to stimulate both frameshifting and stalling of the ribosomes. However because so few

experiments have been performed there is no statistical basis for these results and they merely point

towards a tendency.

7. Discussion

7.1. Modeling the inserted constructs The sequence following the part of the construct predicted to form a stem-loop was designed to prevent

forming of stem2 in 22a/rev6revU, but not in 22a in which the predicted stem-loop structure is followed by

a sequence from the reporter plasmid. Due to this and the similar properties between the supposed stem-

loops and the 22/6a pseudoknot, I designed the most likely RNA structure of the inserted sequences. The

models, based on minimum free energy, were obtained by using the pknotRG web based software (see

references). As seen from fig. 11, the expected stem-loop structures were predicted to form a pseudoknot

structure with approx. the same stability as the 22/6a pseudoknot, the only difference being the length of

stem 2. The most likely structure of the inserted construct from the pTH421 plasmid (fig. 4) was not a

23

pseudoknot and the stability was much lower. This is in agreement with the low frameshift efficiency this

construct exhibited.

In order to find a construct still similar to the 22/6a pseudoknot but more likely to fold as a stem-loop, 4

bases in loop 1 of 22a and 22a/rev6revU were replaced by A’s (fig.12). The inserted sequence from

pSSH1aaaa formed a small pseudoknot while the inserted sequence from pSSH2aaaa was predicted by the

pknotRG software to form stem-loop structures and the stability of the structure was equal to the stability

of the 22/6a pseudoknot. With the same stability and stem1 length as the 22/6a pseudoknot, the stem-

loop forming construct from pSSH2aaaa would be interesting for the future studies of the ability of stem-

loop structures to stimulate ribosomal frameshifting and stalling.

Figure 11: RNA structure of 122 bases starting from the beginning of HindIII site. Modulated based on minimum free energy. pJT538, pSSH1 and pSSH2. (pknotRG software).

24

Figure 12: RNA structure of 122 bases starting from the beginning of HindIII site. Modulated based on minimum free energy. pTH421, pSSH1aaaa and pSSH2aaaa. (pknotRG software)

The fact that the two inserted constructs from pSSH1 and pSSH2 were as likely to be pseudoknots as the

22/6a pseudoknot from pJT515, prevents me from concluding anything concerning the ability of stem-loop

structures to induce frameshift or stall ribosomes. In light of these theoretical models, I find it reasonable

to discuss the results in context of both stem-loop structures and pseudoknots as frameshift signals.

It is however debatable whether it makes sense to understand RNA structures as specific fixed structures,

as these structures probably exist as dynamic structures ‘slipping’ in and out of different favorable

structures. Especially under these conditions where the IPTG induction caused a high expression of the

gene, resulting in ribosomes continuously unwinding the structure.

7.2. The inserted constructs were able to stimulate frameshift In order to examine whether the stem-loop structures stimulate frameshift, the reporter plasmids with

different inserted constructs were transformed into MAS 90 and the bacteria were subjected to a

frameshift assay. I found that the positive control construct 22/6a (pJT515) had a frameshift efficiency of

1.5. In comparison to the frameshift efficiency of 22/6a obtained by Tholstrup et al. (2012) under 1, 1.5 was

slightly higher than expected. A frameshift efficiency of 1.5 is however still lower than the frameshift

efficiency of the other pseudoknots analyzed by Tholstrup et al. (2012). Furthermore, the negative control

(pTH421) was found to have a frameshift efficiency of 0.6. In addition it was ascertained that both expected

25

stem-loop structures (22a and 22a/rev6revU) were able to stimulate frameshift, with a frameshift efficiency

of approx. 1.4 %.

Results in the context of stem-loop frameshift signals: Assuming that the inserted constructs fold into

stem-loop structures, the results are consistent with the finding that an antisense oligo base pairing to the

mRNA, somewhat comparable to a stemloop structure, is able to mimic a frameshift signal (Yu et al. 2010)

and further in agreement with the observation that the position of the paused ribosome seems to be the

same whether its paused by a stem-loop or a pseudoknot (Kontos et al. 2001).

In contradiction to this, other studies show the properties of the stem-loop to be less like the pseudoknot.

Green et al. (2008) found that more mechanical force is needed to unfold the pseudoknot compared to the

stem-loop and that, despite the similarities in thermodynamic stability, the unfolding kinetic is slower when

unwinding the pseudoknot. In contrast to my results, Somogyi et al. (2003) find stem-loops to be less

capable of promoting frameshift compared to pseudoknots.

Results in the context of pseudoknot frameshift signals: As mentioned above, the pknotRG software

calculated, based on minimal free energy, that the constructs could be folded as pseudoknots. Pseudoknot

induced frameshift is well known throughout the literature (reviewed by Giedroc et al. 2009 and Farabaugh

et al. 1996) so if the sequences fold as pseudoknots, this result is not surprising. Somewhat surprising is the

result that the reduced stem 2 length did not reduce frameshift efficiency, as studies have shown that

destabilizing stem 2 reduces frameshift efficiency (Brierley et al. 1991, Kim et al. 1999). If however the

purpose of stem 2 is to increase torsional restraint (Plant et al. 2005), a stem 2 length of 3 base pairs might

prevent rotational freedom to the same extent as a stem 2 length of 6 base pairs. Another study of the

different components of a pseudoknot has shown that reducing stem 2 from 6 to 3 base pairs decreases

the frameshift efficiency from 22 % to 6-3 % (Dam et al.1994). However despite the decrease in frameshift

efficiency, the results of 3-6 % frameshift efficiency prove that a stem 2 length of 3 base pairs is sufficient to

promote frameshift.

As previously mentioned, the length of stem 1 is very important for frameshift efficiency (Napthine et al.

1999). Assuming that stem 1 length is most important for frameshift efficiency, it is not surprising that the

analyzed structures and the positive control had similar frameshift efficiencies, since they all have a stem 1

length of 22 base pairs.

26

7.3. The inserted constructs were able to stimulate ribosomal stalling In order to study stem-loop induced ribosomal stalling, the labeled proteins, expressed from plasmids

containing the different constructs, were separated on nonequilibrium two dimensional gels. The results

from this assay were consistent with the results from the frameshift assay. It was seen that both expected

stem-loop structures were able to stall the ribosome to the same degree as the positive control (22/6a).

This mean that the obtained frameshift efficiency addressed above is lower than actual frameshift

efficiency.

Results in the context of stem-loop frameshift signals: If the analyzed constructs fold into stem-loop

structures, the result that they stimulate ribosomal stalling, is in agreement with the finding that an 18 base

pair stem-loop placed in the 5’ UTR prevents the eukaryotic ribosome from reading through (Kozac 1986).

Also Tsuchihashi et al. (1991) found that a stem-loop structure from E.coli dna-x could lead to ribosomal

pausing. Stem-loop induced pausing of the ribosome has also been observed by Somogyi et al. (1993) and

Kontos et al. (2001) but not for an extended time period. In contradiction, ribosomes have been seen to

bypass stem-loop structures without any delay (Sørensen et al. 1989, Lingelbach et al. 1988).

Results in the context of pseudoknot frameshift signals: Assuming the constructs fold into pseudoknots,

the result, that the analyzed structures were able to promote ribosomal stalling, is consistent with the

results obtained by Tholstrup et al. (2012). My results are furthermore supported by the observations that

pseudoknots promote ribosomal pausing (Somogyi et al. 1993, Kontos et al. 2001) and that the amount of

produced protein decreases when a pseudoknot is located in the reading frame (Plant et al. 2010).

However, as mentioned earlier, other studies have shown that the ribosome can read through larger mRNA

structures without problems (Sørensen et al. 1989, Lingelbach et al. 1988).

8. Conclusion The purpose of this study was to investigate whether a simple stem-loop structure is able to stimulate

ribosomal frameshifting and/or stalling. It is uncertain if the analyzed constructs fold as stem-loop

structures or as pseudoknot structures. In order to conclude how the constructs fold, further testing, for

example NMR spectroscopy, is necessary. However my results suggest that the inserted sequences (22a

and 22a/rev6revU) are able to promote both frameshift and ribosomal stalling. Nevertheless it is important

to keep in mind that my results are based on very little data and therefore lack statistical basis, so at most

my results points towards a tendency.

27

9. Future studies: A downstream stop version of the constructs used in this study: To further study the stalling ability of the

analyzed structures, making versions of the two structures with a stop codon downstream of the frameshift

signal would be interesting. This would force all the ribosomes through the frameshift signal and show a

more distinct stalling tendency. The constructs could be made by removing the upstream stop codon, using

mutational PCR with primers having an AAA codon instead of an UAA stop codon (the last 3 bases in the

spacer). As I am in possession of the plasmid with a UAA to AAA mutant version of 22/6a (pJT521) from the

research of Tholstrup et al. (2012) an alternative method is possible. Digesting my construct with a

restriction enzyme that cuts the construct after the stop codon, but before the bases in loop 2 taking part

of stem 2(Pml I), and with another restriction enzyme that cuts the plasmid outside the construct (PstI) and

ligating it to pJT521 cut with the same restriction enzymes, would do the job. Finding an enzyme with a

unique cutting site at that position proved to be possible with 22a, but not with 22a/rev6revU. Removing

the upstream stop codon extends the reading frame to a stop codon downstream of the inserted construct.

A construct more likely to form stem-loop structures: As this study did not lead to any conclusions with

respect to the frameshift efficiency and stalling ability of stem-loop structures, it would be very interesting

to conduct the same experiments with the proposed 22a/rev6revUaaaa construct. To further study this

construct altering the stability and length of the stem1 could be a possible approach.

28

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