Molecular Biochemistry II

tRNA & Ribosomes

Contents of this page:
Review of nucleic acid structures
tRNA
Amino Acyl tRNA Synthetases
Ribosome Structure

Note to RPI students: Familiarity with basic concepts of molecular biology is assumed. These include the nature of the genetic code, the maintenance of genes through DNA replication, the transcription of information from DNA to mRNA, and the translation of mRNA into protein. Our discussion will start with the material on RNA structure. Basic information is summarized in the first part of these notes, and is covered in more detail in Chapters 29-31 and pages 1285-1291 of Biochemistry, 3rd Edition by Voet & Voet.

Nucleic acids are polymers of nucleotides. Each nucleotide includes a base, either a purine (adenine, guanine) or a pyrimidine (cytosine, uracil,  thymine). See diagram at right and Table 5-1 p. 86. Some nucleic acids contain modified bases. Examples of these are given at right and on p. 1294.
In a nucleotide, e.g., adenosine monophosphate (AMP), the base (e.g., adenine) is bonded to a ribose sugar, which in turn has a phosphate in ester linkage to the 5' hydroxyl, as shown at right.
Nucleic acids have a backbone of alternating phosphate and ribose moieties. Phosphodiester linkages form as the 5' phosphate of one nucleotide forms an ester linkage with the 3' hydroxyl of the adjacent nucleotide in the chain. A short stretch of RNA is shown at right.

Hydrogen bonds link two complementary nucleotide bases on separate nucleic acid strands, or on complementary portions of the same strand. The conventional base pairs are between A & U (or T) and between C & G, as shown in Fig. 5-12 p.88 and below. 

In the diagram at left, H-bonds are shown in red. Relative bond lengths are inexact. 

The image at right is based on X-ray crystallography of tRNAGln (PDB file 1GTR). H atoms are not shown.

Secondary structure: Base pairing over extended stretches of complementary base sequences in two nucleic acid strands stabilizes secondary structure, such as the double helix of DNA (p. 87, 1108). Stacking interactions between adjacent hydrophobic bases in the double helix, contribute to stabilization of such secondary structures. Each base interacts with its neighbors above and below, in the ladder-like arrangement of base pairs in the double helix, e.g., of DNA. 

The genetic code is based on the sequence of bases along a nucleic acid. The standard genetic code is summarized in the Table below. Each codon, consisting of a sequence of 3 bases in mRNA, codes for a particular amino acid, or for chain termination. Some amino acids are specified by two or more codons. Synonyms (multiple codons specifying the same amino acid) in most cases differ only in the third base. Similar codons tend to code for similar amino acids (e.g., codons for the two acidic amino acids Asp & Glu). Thus effects of mutation are minimized.

1st base

2nd base

3rd base

U

C

A

G

U

UUU Phe UCU Ser UAU Tyr UGU Cys

U

UUC Phe UCC Ser UAC Tyr UGC Cys

C

UUA Leu UCA Ser UAA Stop UGA Stop

A

UUG Leu UCG Ser UAG Stop UGG Trp

G

C

CUU Leu CCU Pro CAU His CGU Arg

U

CUC Leu CCC Pro CAC His CGC Arg

C

CUA Leu CCA Pro CAA Gln CGA Arg

A

CUG Leu CCG Pro CAG Gln CGG Arg

G

A

AUU Ile ACU Thr AAU Asn AGU Ser

U

AUC Ile ACC Thr AAC Asn AGC Ser

C

AUA Ile ACA Thr AAA Lys AGA Arg

A

AUG Met* ACG Thr AAG Lys AGG Arg

G

G

GUU Val GCU Ala GAU Asp GGU Gly

U

GUC Val GCC Ala GAC Asp GGC Gly

C

GUA Val GCA Ala GAA Glu GGA Gly

A

GUG Val GCG Ala GAG Glu GGG Gly

G

*Met also codes for initiation.

The genetic code is read during translation via adapter molecules, tRNAs (transfer RNAs) that have 3-base anticodons complementary to codons in mRNA. 

"Wobble" during reading of the mRNA allows some tRNAs to read multiple codons that differ only in the third base (p. 1307-1308). There are 61 codons specifying 20 amino acids. Minimally 31 tRNAs are required for translation, not counting the tRNA that codes for chain initiation. Mammalian cells produce more than 150 tRNAs.

RNA structure: 

Most RNA molecules usually have secondary structure, consisting of stem and loop domains.

  • Double helical stem domains arise from base pairing between complementary stretches of bases within the same strand. These stem structures are stabilized by stacking interactions as well as base pairing, as in DNA.
  • Loop domains occur where lack of complementarity, or the presence of modified bases, prevents base pairing.
The "cloverleaf" model of tRNA emphasizes the two major types of secondary structure, stems and loops.  Transfer RNAs typically include many modified bases, particularly in loop domains.

RNA tertiary structure depends on interactions of bases at distant sites.

  • These interactions generally involve non-standard base pairing and/or interactions involving three or more bases. 
  • Unpaired adenosines (not involved in conventional base pairing) predominate in participating in non-standard interactions that stabilize tertiary RNA structures.
tRNAs have an L-shaped tertiary structure. At right is the structure of tRNAPhe, determined by crystallographic analysis (PDB file1TRA). See also p. 1295-1296.

Extending out from the "acceptor stem," the 3' end of each tRNA has the sequence CCA. The appropriate amino acid is attached to the ribose of the terminal adenine residue (A) at the 3' end of the tRNA, shown in red.

The loop with the anticodon is at the opposite end of the L shape.

An example of non-standard H bond interactions that help to stabilize the L-shaped tertiary structure of a tRNA is shown in ball & stick (left) and spacefill (right) displays (based on Nucleic Acid Database file 1TN2). H atoms are not shown. For other examples see p. 1118, 1296.

Some other RNAs, including viral RNAs and segments of ribosomal RNA, fold in pseudoknots, tertiary structures that mimic the 3D structure of tRNA. Pseudoknots are similarly stabilized by non-standard H-bond interactions.

Explore tRNAPhe (PDB file 1TRA) with Chime.

Right click and change the display to sticks.
Drag the image to visualize the base pairing that stabilizes the secondary structure of stem domains.
Note the double helical structure of stem domains. See how the bases stack on top of one another.

Shift-drag to zoom in for a closer look.
Note the location of the phosphodiester backbone at the periphery of double helical domains.

Identify the acceptor stem, ending in residue #76.

To view one of the more unusual modified bases, designated Y, select hetero-ligand and change the display, e.g., to ball & stick.
This highly modified guanine is adjacent to the anticodon.

Mg++ stabilizes the tertiary structure of tRNA, whose phosphodiester backbone includes multiple negative charges. Hydrated Mg++ ions may contribute to charge compensation at the surface of an RNA molecule. (Note that in a crystal structure not all water molecules may be resolved.)
Look for examples where waters of hydration may be displaced, e.g., when chelated Mg++ interacts directly with RNA phosphate oxygen atoms.


 C
O N S P Mg

Aminoacyl-tRNA Synthetases catalyze linkage of the appropriate amino acid to each tRNA. The 2-step reaction is summarized at right and below.
  1. amino acid + ATP aminoacyl-AMP + PPi
  2. aminoacyl-AMP + tRNA aminoacyl-tRNA + AMP

In step 1, an oxygen atom of the amino acid a-carboxyl attacks the phosphorous of the initial phosphate of ATP.
In step 2, the 2' or 3' hydroxyl of the terminal adenosine of tRNA attacks the amino acid carbonyl carbon atom.

This 2-step reaction catalyzed by Aminoacyl-tRNA Synthetases is spontaneous overall, because the concentration of PPi is kept low by its hydrolysis, catalyzed by Pyrophosphatase.

There is generally a different Aminoacyl-tRNA Synthetase (aaRS) for each amino acid. Accurate translation of the genetic code depends on attachment of each amino acid to an appropriate tRNA. Each aaRS recognizes its particular amino acid and tRNAs coding for that amino acid.

Domains of tRNA recognized by an aaRS are called identity elements (see Fig. 32-14 p. 1299). Most identity elements are located in the acceptor stem and anticodon loop. Aminoacyl-tRNA Synthetases arose early in evolution. Early aaRSs probably recognized tRNAs only by their acceptor stems.

There are two families of Aminoacyl-tRNA Synthetase (aaRSs), designated Class I & Class II. Two different ancestral proteins evolved into the two classes of aaRS enzymes, which differ in the architectures of their active site domains. They bind to opposite sides of the tRNA acceptor stem, resulting in aminoacylation of a different hydroxyl of the tRNA. See also Fig. 32-20 p. 1303.

Class I aaRSs (e.g., GlnRS): Identity elements usually include residues of the anticodon loop as well as the acceptor stem. Class I aaRSs aminoacylate the 2'-OH of the adenosine at their 3' end.

Class II aaRSs: Identity elements for some Class II enzymes do not include the anticodon domain. Class II aaRSs aminoacylate the 3'-OH of the adenosine at their 3' end.

Explore at right an Aminoacyl-tRNA Synthetase with its bound substrate tRNAGln.


GlnRS-tRNAGln

Proofreading/quality control:

Some amino acids are modified after being linked to a tRNA.

  • Some enzymes contain the unusual amino acid selenocysteine (Sec), with selenium substituting for the sulfur atom in cysteine.
    There is a selenocysteine tRNA that differs from other tRNAs, e.g., in having a slightly longer acceptor stem and a unique modified base in the anticodon loop.
    This tRNASec is first loaded with serine via Seryl-tRNA Synthetase. The serine moiety is then converted to selenocysteine by another enzyme, in a reaction involving selenophosphate.
    Sec-tRNASec utilization during protein synthesis requires special elongation factors because the codon for selenocysteine is UGA, which normally is a stop codon.

Other roles of Aminoacyl-tRNA Synthetases:

Ribosomes

Below is a summary of the composition of E. coli ribosomes and mammalian cytoplasmic ribosomes. (See also p. 1310, 1317.) S values refer to sedimentation coefficients. Eukaryotic cytoplasmic ribosomes are larger and more complex than prokaryotic ribosomes. Eukaryotic mitochondrial and chloroplast ribosomes differ from both of the examples shown.

Ribosome Source Whole Ribosome

Small Subunit

Large Subunit

E. coli 70S
  • 30S
  • 16S RNA
  • 21 proteins
  • 50S
  • 23S & 5S RNAs
  • 31 proteins
Rat cytoplasm 80S
  • 40S
  • 18S RNA
  • 33 proteins
  • 60S
  • 28S, 5.8S, & 5S RNAs
  • 49 proteins
Structures of the large and small subunits of bacterial and eukaryotic ribosomes have been determined, by X-ray crystallography and by cryo-electron microscopy with image reconstruction.

Consistent with predicted base pairing, X-ray crystal structures indicate that ribosomal RNAs (rRNAs) have extensive secondary structure (p. 1311 and at right). 

At right are images of the E. coli ribosome, reconstructed from electron density maps obtained by three-dimensional cryo-electron microscopy.

The large subunit is colored cyan, while the small subunit is pale yellow. At left is a surface view. At right is a cut-away view showing the position of messenger RNA (schematically represented as orange beads), as well as P- and E-site tRNAs (based on x-ray structures, shown in green & yellow). The elongation factor EF-G (to be discussed in the next class) is colored blue. This figure was provided by Dr. Joachim Frank, whose laboratory at the Wadsworth Center, New York State Department of Health, carried out the cryo-EM and 3-dimensional image reconstruction. 

Small ribosome subunit: In the translation complex, the mRNA threads through a tunnel within the small ribosomal subunit. The binding sites for tRNA are in a cleft in the small subunit (see above diagram). The 3' end of the 16S rRNA of the E. coli small subunit is involved in mRNA binding.

The small ribosomal subunit is relatively flexible, assuming different conformations. For example, the 30S subunit of a bacterial ribosome was found by cryo-EM and crystallographic analysis to undergo specific conformational changes when interacting with a translation initiation factor (see notes on protein synthesis).

At right the 30S ribosomal subunit of a thermophilic bacterium is displayed by RasMol in spacefill and ribbons, with rRNA in monochrome and protein chains in different colors. (For details see article by B. T. Wimberly et al., 2000.)

The overall shape of the 30S ribosomal subunit is largely determined by the rRNA. The rRNA predominantly consists of double helices (stems) connected by single-stranded loops. The proteins generally have globular domains as well as long extensions that interact with rRNA and may stabilize interactions between RNA helices.

Large ribosomal subunit: At right is the crystal structure of the large subunit of a bacterial ribosome. See also diagrams p. 1314-1316.

The interior of the large subunit is mostly RNA.

Proteins are distributed mainly on the surface. Some proteins have long tails that extend into the interior of the complex. These tails, which are highly basic, interact with the negatively charged RNA.

The active site domain for peptide bond formation is essentially devoid of protein. The peptidyl transferase function is attributed to the 23S rRNA, making this RNA a "ribozyme."

Protein synthesis takes place in a cavity within the ribosome, between the small and large subunits. Nascent polypeptides emerge through a tunnel in the large ribosome subunit. The tunnel lumen is lined with rRNA helices and some ribosomal proteins.

Catalysis of protein synthesis and movement of the ribosome relative to messenger RNA are accompanied by changes in ribosome conformation. Cryo-electron microscopy and X-ray crystallographic studies, carried out in the presence and absence of initiation and elongation factors as well as inhibitors of protein synthesis, have revealed conformational changes in rRNA. Thus rRNA participates in conformational coupling in addition to its structural and catalytic roles.

tRNAs also undergo substantial conformational changes within their ribosomal binding sites during protein synthesis.

 

Explore at right the ribosome large subunit. The data file is very large, so your computer may be slow to respond to commands.

 

Copyright 1998-2008 by Joyce J. Diwan. All rights reserved.



Ribosome Large Subunit
 

Additional material on tRNA & Ribosomes:
Readings, Test Questions & Tutorial

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