Molecular Biology
Familiarity with basic concepts is assumed, including: nature of the genetic code maintenance of genes through DNA replication transcription of information from DNA to mRNA translation of mRNA into protein.
DNA
mRNA
protein
N H
N H
adenine (A)
N H
N H
adenine (A)
O
+
CH3 N
NH2 N N H O
+
O CH3 HN N H NH O
HN H2N N
N H
N H
In a nucleotide, e.g., adenosine monophosphate (AMP), the base is bonded to a ribose sugar, which has a phosphate in ester linkage to the 5' hydroxyl.
NH2 N N N N N HO
5' CH2 4'
NH2
adenine
N N
NH2 N
N H
N
2
N O CH2 H H OH O H
O 3P
O H 1'
3' 2'
ribose adenine
H OH
H OH
H OH
adenosine
Nucleic acids have a backbone of alternating Pi & ribose moieties. Phosphodiester linkages form as the 5' phosphate of one nucleotide forms an ester link with the 3' OH of the adjacent nucleotide. A short stretch of RNA is shown.
NH2
adenine
N
5'
N NH2
5' end O O P O
N O CH2
4'
cytosine
O H 1'
2'
H O P O
3'
ribose
H O
H OH
5'
CH2 H H
3'
O H H OH O
ribose
O O P O
(etc)
3' end
nucleic acid
cytosine (C)
H O
N NH N H N N O H
C G
G C base pair in tRNA
guanine (G)
N N H N
Hydrogen bonds link 2 complementary nucleotide bases on separate nucleic acid strands, or on complementary portions of the same strand. Conventional base pairs: A & U (or T); C & G. In the diagram at left, H-bonds are in red. Bond lengths are inexact. The image at right is based on X-ray crystallography of tRNAGln. 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. Stacking interactions between adjacent hydrophobic bases 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.
Genetic code
The genetic code is based on the sequence of bases along a nucleic acid. Each codon, a sequence of 3 bases in mRNA, codes for a particular amino acid, or for chain termination. Some amino acids are specified by 2 or more codons. Synonyms (multiple codons for the same amino acid) in most cases differ only in the 3rd base. Similar codons tend to code for similar amino acids. Thus effects of mutation are minimized.
Genetic Code
1st base U UUU Phe U UUC Phe UUA Leu UUG Leu CUU Leu C CUC Leu CUA Leu CUG Leu AUU Ile A AUC Ile AUA Ile AUG Met* GUU Val G GUC Val GUA Val GUG Val *Met and initiation. 2nd base C UCU Ser UCC Ser UCA Ser UCG Ser CCU Pro CCC Pro CCA Pro CCG Pro ACU Thr ACC Thr ACA Thr ACG Thr GCU Ala GCC Ala GCA Ala GCG Ala A UAU Tyr UAC Tyr UAA Stop UAG Stop CAU His CAC His CAA Gln CAG Gln AAU Asn AAC Asn AAA Lys AAG Lys GAU Asp GAC Asp GAA Glu GAG Glu G UGU Cys UGC Cys UGA Stop UGG Trp CGU Arg CGC Arg CGA Arg CGG Arg AGU Ser AGC Ser AGA Arg AGG Arg GGU Gly GGC Gly GGA Gly GGG Gly 3rd base U C A G U C A G U C A G U C A G
tRNA
The genetic code is read during translation via adapter molecules, tRNAs, 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 3rd base. 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 have secondary structure, consisting of stem & loop domains.
A : U
U : A
A : U
C : G
C UG C U : G U C U
stem
loop
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.
anticodon loop
tRNA
acceptor stem
The cloverleaf model of tRNA emphasizes the two major types of secondary structure, stems & loops. tRNAs typically include many modified bases, particularly in loop domains.
anticodon loop
Extending from the acceptor stem, the 3' end of each tRNA has the sequence CCA.
anticodon
tRNA
acceptor stem
The appropriate amino acid is attached to the ribose of the terminal adenosine (A, in red) at the 3' end. The anticodon loop is at the opposite end of the L shape.
tRNA
Phe
acceptor stem
#13 C
An example of non-standard H bond interactions that help to stabilize the L-shaped tertiary structure of a tRNA, in ball & stick & spacefill displays.
H atoms are not shown. (From NDB file 1TN2).
Some other RNAs, including viral RNAs & 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 with Chime (PDB file 1TRA).
anticodon
tRNA
Phe
acceptor stem
O R H C C O
O O P O O
O P O O
O P O H O CH2 H OH Adenine O H H OH
NH3+
Amino acid
O R H C NH2 C
O O P O O
ATP
CH2 H H OH
Adenine O H H OH
PPi
Aminoacyl-AMP
Aminoacyl-tRNA Synthetases catalyze linkage of the appropriate amino acid to each tRNA. The reaction occurs in 2 steps. In step 1, an O atom of the amino acid a-carboxyl attacks the P atom of the initial phosphate of ATP.
O R H C NH2 C O
O P O H O CH2 H OH Adenine O H H OH
Aminoacyl-AMP
In step 2, the 2' or 3' OH of the terminal adenosine of tRNA attacks the amino acid carbonyl C atom.
tRNA O O P O H O
tRNA AMP
CH2 H O C HC
3
Adenine O H
2
H OH
O R
Aminoacyl-tRNA
NH3+
Aminoacyl-tRNA Synthetase - summary: 1. amino acid + ATP aminoacyl-AMP + PPi 2. aminoacyl-AMP + tRNA aminoacyl-tRNA + AMP The 2-step reaction 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 & tRNAs coding for that amino acid. Identity elements: tRNA domains recognized by an aaRS. Most identity elements are in the acceptor stem & anticodon loop. Aminoacyl-tRNA Synthetases arose early in evolution.
anticodon loop
tRNA
acceptor stem
tRNA O O P O O
Adenine O H
2
H OH
O R
NH3+
Aminoacyl-tRNA
Two different ancestral proteins evolved into the 2 classes of aaRS enzymes, which differ in the architecture of their active site domains. They bind to opposite sides of the tRNA acceptor stem, aminoacylating a different OH of the tRNA (2' or 3').
Class I aaRSs: Identity elements usually include residues of the anticodon loop & acceptor stem. Class I aaRSs aminoacylate the 2'-OH of adenosine at their 3' end.
Class II aaRSs: Identity elements for some Class II enzymes do not include the anticodon domain. Class II aaRSs tend to aminoacylate the 3'-OH of adenosine at their 3' end.
Proofreading/quality control: Some Aminoacyl-tRNA Synthetases are known to have separate catalytic sites that release by hydrolysis inappropriate amino acids that are misacylated or mistransferred to tRNA. E.g., the aa-tRNA Synthetase for isoleucine (IleRS) a small percentage of the time activates the closely related amino acid valine to valine-AMP. After valine is transferred to tRNAIle, to form Val-tRNAIle, it is removed by hydrolysis at a separate active site of IleRS that accommodates Val but not the larger Ile. In some bacteria, editing of some misacylated tRNAs is carried out by separate proteins that may be evolutionary precursors to editing domains of aa-tRNA Synthetases.
Some amino acids are modified after being linked to a tRNA. Examples: In prokaryotes the initiator tRNAfMet is first charged with methionine. Methionyl-tRNA formyltransferase then catalyzes formylation of the methionine, using tetrahydrofolate as formyl donor, to yield formylmethionyl-tRNAfMet. In some prokaryotes, a non-discriminating aaRS loads aspartate onto tRNAAsn. The aspartate moiety is then converted by an amidotransferase to asparagine, yielding Asn-tRNAAsn. Glu-tRNAGln is similarly formed and converted to Gln-tRNAGln in such organisms.
Some proteins contain the unusual amino acid selenocysteine (Sec), with selenium substituting for the sulfur atom in cysteine. The tRNASec is first loaded with serine via Seryl-tRNA Synthetase. The serine moiety is then converted to selenocysteine by another enzyme. Utilization of Sec-tRNASec during protein synthesis also requires special elongation factors.
Proteolytic cleavage of the human aaRSTyr yields a cytokine that stimulates angiogenesis. A truncated form of the human aaRSTrp inhibits angiogenesis. Regulation of apoptosis by the human aaRSGln is dependent on the concentration of its substrate glutamine. Several mammalian Aminoacyl-tRNA Synthetases associate with other proteins to form large macromolecular complexes whose roles are actively being investigated.
Rat cytoplasm
80S
Eukaryotic cytoplasmic ribosomes are larger and more complex than prokaryotic ribosomes. Mitochondrial and chloroplast ribosomes differ from both examples shown.
5S rRNA
crown view displayed as ribbons & sticks.
PDB 1FFK
Structures of large & small subunits of bacterial & eukaryotic ribosomes have been determined, by X-ray crystallography & by cryo-EM with image reconstruction. Consistent with predicted base pairing, X-ray crystal structures indicate that ribosomal RNAs (rRNAs) have extensive secondary structure.
EF-G
small subunit
mRNA location
The cutaway view at right shows positions of tRNA (P, E sites) & mRNA (as orange beads). EF-G will be discussed later. This figure was provided by Joachim Frank, whose lab at the Wadsworth Center carried out the cryo-EM and 3D image reconstruction on which the images are based.
Small ribosomal subunit of a thermophilic bacterium: rRNA in monochrome; proteins in varied colors.
PDB 1FJF
spacefill display
ribbons
The overall shape of the 30S ribosomal subunit is largely determined by the rRNA. The rRNA mainly 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.
The active site domain for peptide bond formation is essentially devoid of protein. Peptidyl transferase is attributed to 23S rRNA, making this RNA a "ribozyme." A universally conserved adenosine base serves as a general acid base during peptide bond formation.
PDB 1FFK
Protein synthesis takes PDB 1FFK place in a cavity within the ribosome. Nascent polypeptides emerge through a tunnel in the large subunit. Some nascent proteins then pass through a channel into the ER lumen, or across the cytoplasmic membrane Large ribosome subunit. Backbone display with RNAs blue. View and out of the cell in from bottom at tunnel exit. prokaryotes.
small subunit
large subunit
Sec61 channel
The cutaway view at right shows that the tunnel in the yeast large ribosome subunit, through which nascent polypeptides emerge from the ribosome, lines up with the lumen of the ER Sec61 channel.
Figure provided by Joachim Frank, whose lab carried out the cryo-EM & image reconstruction on which these images are based.