Protein biosynthesis:transfer RNA (tRNA)

05.09.07Rya Ero

General reminders• tRNAs are adaptor molecules for translating

nucleotide sequence of mRNA into amino acid sequence of proteins

• deciphrering via genetic code• 30–40 different tRNAs identified in bacterial

cells and as many as 50–100 in animal and plant cells

Structural properties of RNA• RNA vs. DNA - polynucleotide

chains with ribose sugar insead of deoxyribose & uracil vs. thymine

• 2’ hydroxyl of ribose enables more tertiary interactions, destabilize the 5’-3’phosphodiester bonds and prevent formation of B form double helix

• RNAs single stranded molecules, often fold onto themselves (tRNA, rRNA)

Structure of tRNA• Similar overall structure vs. unique identifying

sequences • Pioneer molecule in structural studies of RNA• Existence was predicted by Francis Crick in

1956• Sequenced in 1965 by Robert Holley, secondary

structure prediction• 1976 Kim and Rich and Klug and colleagues

describe the detailed three-dimensional structure of tRNA determined by x-ray diffraction.

Primary and secondary structure• 60-95 nt (18-28 kDa),

commonly 76 nt long• uniform numbering

(anticodon 34-36) • 5’ terminus phosphorylated

(pG)• 3’ CCA-OH (74-76)• conserved, semi-conserved

and variable positions • extensively modified (7-15

per molecule) (>100 all together)

• cloverleaf-like secondary structure

• A-form double- stranded helices

• intramolecular hydrogen bonding and extensive stacking interactions

• non-Watson-Crick base pairs

• 4 “arms”• “extra aka variable arm”

(up to 7bp) 3-21 nt

• most invariant and semi-invariant (either purine or pyrimidine) positions in loop regions

The conserved nucleotide residues in tRNA are: 8, 11, 14, 15, 18, 19, 21, 24, 32, 33, 37, 48, 53, 54, 55, 56, 57, 58, 60, 61, 74, 75, 76.

D-loop anticodon loop T-loop acceptor stem

• 15 invariant nucleotides: 5' p

*U

Y

R

AR

*GG A

Y

U R*

G

C

T UC

RA*Y

CCA - OH 3'

Y*

anticodon loop

amino acid acceptor stem

T-loopD-loop

variable arm

1

76

often I

• 8 semi variant nucleotides: 5' p

*U

Y

R

AR

*GG A

Y

U R*

G

C

T UC

RA*Y

CCA - OH 3'

Y*

anticodon loop

amino acid acceptor stem

T-loopD-loop

variable arm

1

76

often I

• regions of lengthvariation ------

5' p

*U

Y

R

AR

*GG A

Y

U R*

G

C

T UC

RA*Y

CCA - OH 3'

Y*

anticodon loop

amino acid acceptor stem

T-loopD-loop

variable arm

1

76

often I

• examples of non-Watson-Crick base pairs in tRNA:

• G-U bp in helical regions of tRNA

• A-G, A-A, A-C G-G, and reverse G-C bp take part in cross-linking in the non-helical regions of tRNA.

Tertiary structure• “L–shape” with two continuous arms (coaxial

stacking ) at 90°• amino acid acceptor region and the anticodon

region are about 75 Å apart

• 3D stabilized by base stacking, base intercalation, and additional hydrogen bond formation (non-Watson-Crick bp)

Stacking interactions: aromatic rings of the bases are involved in hydrophobic interactions that stabilize the tertiary structure of tRNA; can be seen in one axis through the acceptor stem and the TΨC loop and in another axis through the anticodon stem and the D-loop.

• base stacking interactions can be seen between the residue 53 to 55 and between the residues 56 to 58 in TΨC loop .

Intercalation: reversible inclusion of a molecule (or group) between two other molecules (or groups), bases, in case of tRNA. In the crystal structure of tRNA, two examples can be seen:

• G57 of TΨC loop intercalates between G18 and G19 of the D-loop.

• A9 of the base of D-stem intercalates between G45 and 7mG46 of the variable loop.

Conserved bases participate in tertiary base pairing interactions that:

• maintain the loop structures• hold the D-loop, the variable loop and the TΨC

loop together

Most of the bases, as well as the phosphate backbone, and the 2’-OH of the ribose of non-helical regions participate in tertiary hydrogen bonding interactions.

Many of the tertiary base pairing interactions in the loop regions are those of non-Watson-Crick base pairs

• following base triples are found in phenylalanine tRNA:G10-C25 base pair & G45;A23-U12 base pair & A9;G22-C13 base pair & G46.

• conserved G19⋅C56 pair locks the nearby-situated D and TΨC loops

• U8⋅A14 reverse Hoogsteen pair stabilizes the sharp turn in the D loop

• tertiary pairs of Gm18⋅Ψ55 and m5U54⋅A58(modified nucleosides)

• Anticodon bases are accessible, allowingbase pairing with codons in mRNA.

• The invariant purine on the 3' side of the anticodon is typically (hyper)modified (Y37). This modified purine base is also accessible in solution and may assist the anticodon-codon interaction.

• Sharp turn of the tRNA backbone occurs following conserved U33 in front of the anticodon (U-turn)

• stabilized through interactions between U33 and the phosphate oxygen three residues downstream toward 3' terminus (P36).

• U-turn also occurs after Ψ55 in the TΨC loop.

• stabilized byinteractions between Ψ55 and P58.

• phosphate oxygen two residues down stream is positioned directly beneath the base

• Mg2+ ions can associate with the phosphate backbone of tRNA

• stabilize 3D structureof tRNA and may assist tRNA folding.

• In the crystal structure, four Mg2+ binding sites can be seen.

• Mg2+ ion in the pocket of D-loop is coordinated by 6 water molecules, which in turn participate in hydrogen bonding interactions with phosphate oxygens. Mg2+ ions in other sites are directly coordinated by 1 or 2 phosphate oxygens and the remaining 4 or 5 sites are occupied by water molecules, which participate in hydrogen bonding interactions with nitrogens or oxygens of bases.

• polyamines (especially spermidine) play an important role

Overview of tRNA structure

• See tutorial: http://www3.interscience.wiley.com:8100/legacy/college/boyer/0471661791/structure/tRNA/trna.htm

Biosynthesis of tRNA

• In both bacteria and eukaryotes, tRNA genes occur singly and as multigene transcription units

• synthesised as long precursor molecules (pre-tRNAs)

• pre-tRNAs are trimmed to produce the tRNA of mature size

• several additional processing steps...

Transcription in prokaryotes

• some tRNAs are included in the pre-rRNA transcripts

• cleavage (by ribonuclease III) of this transcript produces 5S, 16S, and 23S rRNA molecules and a tRNA molecule

• other transcripts contain arrays of several kinds of tRNA or of several copies of the same tRNA

Transcription in eukaryotes• transcribed by RNA

polymerase III (also 5S rRNA) in nucleoplasm

• promoter regions of tRNA genes lie entirely within the transcribed sequence

• 2 internal promoter elements, A box and B box, are present in all tRNA genes

Promoters of tRNA genes are downstream of the transcription initiation site and do not contain a binding site for TFIIIA. TFIIIC initiates transcription by binding to promoter sequences, followed by the association of TFIIIB and polymerase. The TATA-binding protein (TBP) is a subunit of TFIIIB (S. cerevisiae )

tRNA maturation

• Generation of mature ends:• processing of the 5’ end involves cleavage by

ribonuclease P (RNase P) • 3’ end is generated by the action of numerous

protein RNases • CCA sequence is often synthesized de novo by

a template independent RNA polymerase, CCA-adding enzyme

• some pre-tRNAs from both bacteria and eukaryotes contain introns that are removed by splicing

• extensive modification of bases • tRNAs generally are associated with

proteins and spend little time free in the cell • export of tRNAs from the nucleus (through

nuclear pore complexes) is a critical step in eukaryotic cells

• protein factors may assist the formation of tRNA tertiary structure

• Aquifex aeolicus Trbp111 is an RNA chaperone

• Processing of Tyr pre-tRNA involves four types of changes. An intron in the anticodon loop is removed by splicing. A 16-nt sequence at the 5′ end is cleaved by RNase P. U residues at the 3′ end are replaced by the CCA sequence. Numerous bases are converted to modified bases.

Generation of mature 3’ and 5’ends

• Primary transcript forms cloverleaf structure (two additional hairpins)

• first cut ribonuclease E or F forming a new 3′.• ribonuclease D (exonuclease) trims 7 nt and

then pauses...• ... while ribonuclease P makes a cut at the start

of the cloverleaf, forming the 5′ end. • ribonuclease D then removes 2 more nt,

creating the mature 3′ end.

In tyrosine tRNA the 3′-terminal CCA sequence is present in the RNA and is not removed by ribonuclease D. With some other tRNAs this sequence has to be completely or partly added by tRNA nucleotidyltransferase

• processing of the 5’ end of pre-tRNAs involves cleavage by ribonuclease P (RNase P)

• catalytic RNA molecule (model for ribozyme) • RNase P consists of RNA and protein

molecules, both of which are required for maximal activity

• isolated RNA component (M1) of RNase P is itself capable of catalyzing pre-tRNA cleavage (1983 Sidney Altman and colleagues)

• the 3’ end of tRNAs is generated by the action of numerous conventional protein RNases

• 3’ CCA essential for amino acid attachment and interaction with the ribosome

• encoded in the DNA of some tRNA genes• often synthesized de novo by a template

independent RNA polymerase, CCA-adding enzyme (nucleotidyl transferase)

• reaction proceeds as follows:• tRNA +CTP --> tRNA-C + PPi • tRNA-C +CTP --> tRNA-C-C + PPi• tRNA-C-C +ATP --> tRNA-C-C-A + PPi

Splicing• some pre-tRNAs from both bacteria and eukaryotes

contain introns, removed by splicing• introns are always located in the anticodon loop• pre-tRNAs are most likely folded similarly to mature

tRNAs, bringing the two intron-exon junctions into proximity

• trimming and splicing reactions are believed to act as quality control steps in generation of functional tRNAs (misfolded tRNA precursors are not processed properly)

• mechanism of pre-tRNA splicing differs from other types of splicing reactions (protein enzymes)

• mechanism of splicing: • first, the pre-tRNA is cleaved at two places,

thereby excising the intron and generating a 2′,3′-cyclic phosphomonoester at the 3′ end of the 5′ exon

• multi step reaction joining the two exons requires two nucleoside triphosphates: a GTP, which contributes the phosphate group for the 3′→5′ linkage in the finished tRNA molecule; and an ATP, which forms an activated ligase-AMP intermediate

• 2′-phosphate on the 5′ exon is removed in the final step

• Splicing of the S.cerevisiae pre-tRNATyr

• 2′,3′-P terminus is converted to a 3′-OH end by a phosphodiesterase

• 5′-OH terminus is converted to 5′-P by a kinase

• these two ends are then ligated together.

• Structure of a tRNA-splicing endonuclease docked to a precursor tRNA. The endonuclease (a four-subunit enzyme) removes the tRNA intron (blue).

tRNA modification

• formed post-transcriptionally by enzymatic modification of standard ribonucleotides in a tRNA precursor

• modification and site specific enzymes• modification site in archaeal tRNA is determined

by snoRNA, modification (Ψ and 2’-O-methylation) synthesis catalyzed by proteins

• Stabilize 3D of tRNA• allow greater structural and functional versatility

of tRNA

• found at various positions in the tRNA • positions 34 and 37 contain the largest variety of

rather complex modifications (hypermodified nucleosides)

• some modifications (Ψ) are found in several positions of tRNA, others have unique location

• some modifications mediate the recognition of tRNAs by aminoacyl-tRNA synhetases

• some increase the range of the interactions that can occur between tRNAs and codons during translation

• methylation of 2’-OH group of the ribose serves to generally stabilize RNA structure

• methylation of nucleosides prevents the formation of usual bp, rendering bases accessible for tertiary interactions

• methylation imparts a hydrophobic character to some regions of tRNA, important for interaction with proteins

• Ψ can form additional hydrogen bond compared to uridine, decrease conformational flexibility

• D has destabilizing effect on RNA structure• Ψ and m5U promote the syn conformation of the

glycoside bond and the 3’ endo conformation of the sugar, improve stacking interactions

• in all tRNA molecules (ubiquitous feature

• about 10% of bases• >100 different modified nucleoside (tRNA, rRNA,

snRNA, snoRNA), most only in tRNA and very rare

• types of modifications:• modification of bases• modification of ribose• pseudouridine (Ψ)

• Methylation - addition of one or more -CH3 groups to the base or sugar (G>m7G)

• Deamination - removal of an amino (-NH2) group from the base (A>I)

• Sulfur substitution -Replacement of oxygen with sulfur (U>t4U)

• Base isomerization -changing the positions of atoms in the ring component of the base(U>Ψ)

• Double-bond saturation - converting a double bond to a single bond (U>DHU)

• Nucleotide replacement - replacement of an existing nucleotide with a new one (>Q)

4-thiouridine (S 4U)

CN C

C CN

HH

S

O H

Ribose

Pseudouridine ( ψ )

CN

C C

H

O H

Ribose

O

c

N

HC

N C

C CN

H

O H

Ribose

O

CH 3

Ribothymidine (T)

CN C

C CN

H

O

Ribose

O

H

HHH

Dihydrouridine (D) 1-Methylguanosine (m G)1

CN C

C CN

Ribose

O

CH 3

NH

H

C

N

N

H

CN C

C CN

Ribose

O

C

N

N

H

H

H

Inosine (I)

CN C

C CN

Ribose

C

N

N

H

CH 2 CH CCH 3

CH 3

H

NH

N isopentenyladenosine (i 66 A)-

• 4 times more genetic information is devoted to the synthesis of modification enzymes, than to the synthesis of tRNA.

• tRNA modification enzymes are divided into two large groups based on their sensitivity to the tertiary structure of tRNA

• use different mechanisms to recognize their substrate tRNAs

• early enzymes of tRNA processing may facilitate and/or stabilize the folding of tRNA precursors into correct tertiary structure

• Pseudouridine (Ψ) is most common modification

TruB

• tRNAs from most organisms have Ψ at position 55

• tRNA-(Ψ55) synthase (TruB) recognizes and modifies all the tRNAs in E. coli cells

• recognition signals for TruB are contained entirely in the TSL region tRNA

• in pre-tRNA the substrate U55 is hydrogen bonded to Gm18 in the D loop and the base is unavailable for TruB

• TruB must disrupt at least partially the tertiary structure of tRNA precursor

(a) tRNA T loop is bound in a cleft of Ψ synthase domain.

(b) Conformation of T loop nt in folded yeast tRNAPhe. 2 nt from the T loop make tertiary contacts with D loop.

(c) Conformation of T loop when bound to TruB. Ψ55, C56 and G57 areflipped out. This allows the TruB to access the substrate base 55, but also disrupts the two bpformed between D and T loop nt.

• Ψ55 not essential• TruB has RNA chaperone activity and is actively

involved in process of tRNA folding (?)• opening of tRNA structure during pseudouridine

synthesis may give to misfolded molecules a chance to acquire correct tertiary structure

• tRNA modification enzymes may act as RNA chaperones

• modification enzymes form large multi-enzyme complexes (?)