Possible relationship of peptidyl transferase binding sites, 5S RNA and peptidyl-tRNA

Vol. 45, No. 3, 1971

BIOCHEMICAL

POSSIBLE RELATIONSHIP

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

OF PEPTIDYL TRANSFERASE BINDING SITES,

5s RNA AND PEPTIDYL-tRNA STANISLAV

CHLADEK

Detroit Institute of Cancer Research, Division of the Michigan Cancer Foundation, Detroit, Michigan 48201 Received

August

23, 1971

SUMMARY A possible role of the 5s RNA in the peptide chain elongation process is suggested. This role consists of an interaction of the Y loop of peptidyl tRNA at the P-site of peptidyl transferase with the single stranded region of the 5'S 'RNA. The interacting region may also be one of the sites for peptidyl transferase binding to the 55 RNA, moreover, the resulting "release" of peptidyl transferase from its binding site on the 5S RNA (due to binding of the Y loop of peptidyl tRNA to the 5S RNA) could cause conformational changes in the enzyme leading to an exposure of its "CCA binding site" for peptidyl tRNA. Peptidyl enzymes)

is

ribosomes

1.

accepted. site

transferase, considered

a peptide to be an integral

The two-binding Aminoacyl

and peptidyl

of peptidyl

site

transfer

prior

tRNAs must

group

of AA-tRNA

at the A-site.

Relatively

simple

(or

units)

interact to act conditions

with

can replace

peptidyl

transferase

as an acceptor have

"short

fragments":

* This

has become

derived whole

or donor*

to be used (a) known

the peptidyl

from

of the peptide

higher

Mg*

as the

fragment

acceptor

concentration reaction. 695

now widely acceptor

site

The acceptor

(CCA)

bond

for chain4.

nucleosides

corresponding

either

and

(b)

of AA-tRNA tRNAs and

A- or P-site,

Nevertheless,

or donor

forma-

to the amino

the 3'-terminus

of the

(A)

(P) or donor

peptide

(or N-acyl-AA)

as substrates

the

is

tRNA at the P-site

molecules

to elicit

2 23 .

of

of bacterial

in the

to accomplish from

complex

transferase

formation

such as AA-

oligonucleotides

N-acyl-AA-tRNA)

bond

(or

505 subunit

to be bound

tRNA in

residue

compounds

of the

believed

be in juxtaposition

the peptide

enzyme

of peptidyl

to peptide

by transferring

(2-6

model

RNA is

tion

or short

part

(or N-acyl-sminoacyl)

transferase

ends of both

bond-forming

activity for

binding

i.e.,

special of the to the

Vol. 45, No. 3, 1971

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

TRANSFERASE

a Fig.

Simplified

1.

(a)

model

substrate It

must be present4.

to the P-site

way4*5. tion

Since

vity those

there

appropriate

of AA (or

peptidyl

recognition

sites

for

or peptide

ribosomal

complex

than

of the

that

of tRNAs in

has been

and 5s RNA binding

5s RNA site (by interaction P-site is accessible for The situation is depicted formation.

concentration

suggested

, perhaps

for

peptidyl

of corresponding

binding

a significant

that

changing

is no requirement

in some instances)

to the

transferase

sites. the

The 5s RNA binds to the peptidyl transferase site, the P-site is then closed for the entry of the peptidyl-CCA derivative (alcohol is not present). Peptide bond formation does not take place.

(donor)

substrate

of peptidyl

Peptidyl-tRNA is locked in of its GTYC$ loop) and the CCA end of peptidyl-tFWA. prior to the peptide bond

(b)

P-site

b

for

ethanol

sites,

which

necessitates

containing

intact

end fragments, to the

It tlWAs

is

CCA sequence, 696

the

it these

in some

u

concentra-

transfer

could

are

actilower

be a lack

modified

generally

binding

of the

tRNA (AA or peptidyl)

assumed

and messenger

due to other

Mg

of fragments

themselves4,

formation.

conformation

and since

derivatives

alcohol

binding

(or higher

transferase

tRNA derivatives

promotes

the ribosomal

of a complete

on fragments

the addition

ethanol

the binding

or N-acyl-AA)

bond

of an aliphatic

than of

conditions that

the

RNA is more stable

sites

in intact

The CCA sequence

is

molecules nevertheless

Vol. 45, No. 3, 1971

considered rectly

as the only

with

peptidyl

BIOCHEMICAL

AND BIOPHYSICAL

recognition

site

common sequence"

far7

and the

binding

suggested

5s RNAI*.

Indeed,

single

stranded

actually

tidy1

to both

the

A- and P-sites*,'.

like

site

to the

conformational

an increase

in the

exposure

molecules.

In other

the P-site,

some closely

on the 50s subunit If

we consider

can further

suppose

transferase. 5S RNA (the binding

sequence

site

for

peptidyl

5S RNA (which conformational

of peptidyl

would

result

changes

in

of the GTYC; sequence be locked

at two main

the enzyme leading

to 55 RNA, we

part

of the

tRNA)

changes

It would at least

of tRNA)

loci

5S RNA and peptidyl

to 55 RNA template

the binding

could

to an exposure

of the is

in

also

can thus then

be

seem possible

one binding cause

it

has been

suggested

that

697

the

ribosomal

site

pronounced

of CCA binding

complex

a

the enzyme

sites. Recently,

to

(anticodon-codon).

of tRNA is

Conformational

from

of tRNA

are normally

single-stranded

effect.

in

transferase

accessible4)

to GTYC:

transferase

from

is

results

of peptidyl

between

the

complementary

as an allosteric-like

the "release"

then

that

roughly

in a

of pep-

the CCA parts

sites"

exists

of tRNAs GTYC: sequence

that

located

This

sites.

of the GTYC: portion

transferase.

to

of the A- or P-site

binding

prebinding

due to the binding classified

possibly

GTYC$ binding

one on the 30s subunit

propose

CGAACll

the GTYC: sequence

that

to be less

relationship

further

idea

these

for

The tRNA would

a close

I would

sites

may require

and an additional

non-enzymatic

seem unreasonable.

in

is known

the binding

to inhibit

the neighborhood

changes

which

site.

that

the

the "CCA binding

exposure

related

in

of both

words,

shown

of the GTYC: sequence

binding

occurs

causes

and their

and thus

that

and further

dormant

di-

tRNAs sequenced

of CGAAC has been

5s RNA does not

to propose

in all

Therefore,

sequence

of 55 IWAll

tRNA (N-acyl-AA-tRNA)

(particularly

to interact

of tRNA to 50s ribosomes,

complementary

region

found

TYCG has been

as a binding

the binding

I would

GTYCt6 has been

tetranucleotide

of AA-tEUA

has been

the tRNA molecule

transferase4.

"The thus

in

RESEARCH COMMUNICATIONS

passes

on

BIOCHEMICAL

Vol. 45, No. 3, 1971

through

a cycle

tion12.

of contraction

could

here,

Even though formational folding

could

site8sq.

while

further

to appropriate

reasonable

that

the

required.

This

is

the same number

if

site) tration),

(for

is

alcohol)

tortion

the

the

supported in

the I loop

of tRNA [like

"CCA locus"

part

"fragment

of the reaction transferase

of substrate of the

from

for where

sites

the A-site

by ethanol,

instead

5 base

could

pairs

assumed

directly

(P-

i-k

concen-

is necessary. On the other the part

of the

(or

substrate.

other

alipha-

of partial

dis-

an uncontrolled take

place

of an enzymatically

under mediated

translocation17. Since one might

the P- and A-sites even suggest

may be considered

conformational

changes

698

as allosterically in one site

tRNA

means be

of the

As a result

to the P-site

reaction"

all

more drastic

ethanol

be

tRNAs have

10 ml4 Mg

the binding

environment.

binding

"fragment

that

seem

can be also

where

of

would

concentration.)

requires

reaction"4

known

P-site

CCA (fMet),

of the P-site

all

about

closed

loop

would

to rebosome (at

ff

it

It

15).

Mg

in helical

and almost

to be bound

by higher

of GTYC:

stem region

that

(7)15

see ref.

un-

in the dhU 100~'~.

AUG triplet),

fact

e.g.,

of anticodon

the adjacent

by the

is

be caused

case of the is

conditions

or even

to 5s RNA is missing]

of peptidyl

diffusion

'4 loop

of normally

binding

This

to coding

distortion

for

part

con-

of the GTYC: segment

the binding

gentle

chain

to expose

with

translocation16

a mere end fragment

to take

the binding

directed

hand,

used

about

G-factor

would

additional

to the A and G residues

an exception,

distortion

model,

considered

response

further

(This

tic

of an exposure

or N-acyl-AA-tRNA

only

the

of tRNA as a result

of nucleotides

peptidyl

without

(in entire

the stem region

that

tRNA molecules,

usually

as

process.

in substrate

From an analogy

to ribosome14

overall

transferase,

occur

speculate

loci.

by peptidyl

of transla-

the proposed

T, Y and C may bind

One could

Met tRN AF

G is

the process

for

also

structure

during

undergone

to the

not essential

of tertiary

pairing

changes

contribute

changes

to a ribosomal

in

and expansion

The conformational

suggested

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

induced

linked, by a sub-

Vol. 45, No. 3, 1971

strate

bound

results

to the second

of Erbe

tide

bond

the

former

duces

could

not,have

P-site

conformational

role

This

to the P-site.

This

is made possible

changes

to the proper

in

site

this

mutual

interactions

which

suggests

linked

with

chain peptidyl

that

binding above)

A-site.

first

elongation

that

transferase

the pep-

bond

unless

of N-acylfurther

AA-tRNA

attempt

process**

the

and AA-tRNA

of a peptide the

to explain

indicate

as suggested

allosterically

constitutes

serve

N-acyl-AA-tRNA

and the formation

model

RESEARCH COMMUNICATIONS

would

et aZ.lq between

of the 5s RNA in the peptide

of its

assumption

formed

(which

To my knowledge

AND BIOPHYSICAL

and Shorey been

was prebound

bound

site.

and Leder'*

AA-tR.NA to the

then

BIOCHEMICAL

inis

takes

to assign

place. the

on the basis

and peptidyl

(N-acyl-MA)

tRNA.

ACKNOWLEDGMENTS I wish to thank Dr. J. Zemlicka of the Michigan Dr. 8. McAllister of Wayne State University for their criticisms.

Cancer helpful

Foundation discussions

and and

REFERENCES Monro,

R. E.,

Properties

6

8 9 10 11 12 13 14 15 16

Maden,

B. E. H.,

and Function (edit.

and Traut, By Shugar,

R. R., in Genetic Elements, D.), 179 (Academic Press,

London, 1967). Watson, J. D., BUZZ. Sot. Chim. Biol., 46, 1399 (1964). Bretscher, M. S., and Marcker, K. A., Nature, 211, 380 (1966). Monro, R. E., Staehelin, T., Celma, M. L., and Vazquez, D., Cold Spring Harbour Symp. Quant. Bioz., 34, 357 (1969) and references therein. Silverstein, E., Biochim. Biophys. Acta, 186, 402 (1969). Zamir, A., Holley, R. W., and Marquisee, M., J. BioZ. Chem., 240 1267 (1965). Zachau, H. G., Angew. Chem. Intemat. Edit., 8, 711 (1969). Ofengand, J., and Henes, C., J. BioZ. Chem., 244, 6241 (1969). Shimizu, N., Hayashi, H., and Miura, K., J. Biochem., 67, 373 (1970). Pestka, S., CoZd Spring Harbour Symp. Quant. BioZ., 34, 395 (1969). Jordan, B. R., J. Mol. BioZ., 55, 423 (1971). Schreier, M. H., and Noll, H., Proc. U. S. National Acad. Sk., 68, 805 (1971). Levitt,

M.,

Nature, 224, 759 (1969).

Clark, B. F. C., Dube, S. K., and Marcker, Philipps, G. S., Nature, 223, 374 (1969). Bretscher, M. S., Nature, 218, 675 (1968).

K. A.,

Nature,

219,

484 (1968).

** Very recently, it has been shown20 that ribosomes reconstituted in the absence of the 5s RNA fail to incorporate 4 (50s) proteins and do not possess peptidyl transferase activity. 699

Vol. 45, No. 3, 1971

I7 I8 19 *O

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Monro, R. E., Nature, 223, 903 (1969). Erbe, R. W., and Leder, P., Biochem. Biophys. Res. Commun.,31, 798 (1968). Shorey, R. L., Ravel, J. M., and Shive, W., Nature, 226, 358 (1970). Erdmann, V. A., Fahnestock, S., and Nomura, M., Fed. Proc., 30, 1203 (1971).

700