Molecular Genetics-223

Spring 2012

nature of the genetic code maintenance of genes through DNA replication transcription of information from DNA to mRNA translation of mRNA into protein DNA repair Recombination Intron splicing

Brenner S., Jacob F., and Meselson M. (1961) An unstable intermediate carrying information from genes to ribosomes for protein synthesis. Nature 190: 576-581
(13 May 1961)

RNA
Composition Structure Function Stability

Chemistry of Information
Nucleic acids are polymers of nucleotides. Each nucleotide includes a nucleoside base that is either a purine (adenine or guanine), or a pyrimidine (cytosine, uracil, or thymine).
Nucleoside bases found in RNA:
NH2 N N N HN H2N N guanine (G) O N NH2 N N H cytosine (C) O N H uracil (U) O NH O

N H

N H

adenine (A)

RNA species may contain modified bases. Examples:

Nucleoside bases found in RNA:
NH2 N N N HN H2N N guanine (G) O N NH2 N N H cytosine (C) O N H uracil (U) O NH O

N H

N H

adenine (A)

Examples of modified bases found in tRNA:

NH2 H3C
+N

O
+

CH3 N

NH2 N N H O
+

O CH3 HN N H NH O

HN H2N N

N H

N H

1-methyladenine (m1A) 7-methylguanine (m7G) 3-methylcytosine (m3C) pseudouracil ()

In a nucleotide, e.g., adenosine monophosphate (AMP), the base is bound 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

adenosine monophosphate (AMP)

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 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.

RNA structure:
Most RNAs have secondary structure, consisting of stem & loop domains. Double helical stems arise from base pairing between complementary stretches of bases within the same strand. Loops occur where lack of complementarity, or the presence of modified bases, prevents base pairing.

anticodon loop

The cloverleaf model of tRNA secondary structure emphasizes the 2 major types of secondary structure, stem and loop domains.

tRNA

acceptor stem

tRNAs typically include many modified bases, particularly in the loop domains.
Tertiary structure depends on interactions of bases at more distant sites. Many of these interactions involve non-standard base pairing and/or interactions involving three or more bases. tRNAs usually fold into an L-shaped tertiary structure.

anticodon loop

Extending out from the "acceptor stem", the 3' end of every tRNA has the sequence CCA.
anticodon

tRNA

acceptor stem

The amino acid attaches to the ribose of the terminal A (in red) at the 3' end.
The anticodon loop is at the opposite end of the L shape.

tRNA

Phe

acceptor stem

Tertiary base pairs

#46 (m7G) #22 G #22 G

#46 (m7G) #13 C

#13 C

Tertiary base pairs in tRNAPhe

Tertiary base Phe pairs in tRNA

Non-standard H bond interactions, some linking 3 bases, help stabilize the L-shaped tertiary structure of tRNA. H atoms are not shown.

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

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 two 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

(terminal 3nucleotide of appropriate tRNA)

O R

Aminoacyl-tRNA

NH3+

Aminoacyl-tRNA Synthetase
Summary of the 2-step reaction: 1. amino acid + ATP aminoacyl-AMP + PPi

2. aminoacyl-AMP + tRNA aminoacyl-tRNA + AMP

There is a different Aminoacyl-tRNA Synthetase (aaRS) for each amino acid. Each aaRS recognizes its particular amino acid and the tRNAs coding for that amino acid. Domains of tRNA recognized by an aaRS are called identity elements. Most identity elements are in the anticodon loop acceptor stem & anticodon loop. Aminoacyl-tRNA Synthetases arose early in evolution
tRNA acceptor stem

One of the current models of translation start

Key enzymes:

1) Guanylyl transferase, 2) Guanine-7-methyltransferase, 3) 2-O-metyltransferase.

An example to viral mechanisms blocking eukaryotic translation

Prevention of cap-dependent translation.

Viral protease (2A-pro) cleaves eIF4G.

Firefly luciferase mRNA (with 5 and 3 UTRs from a plant gene) was electroporated into protoplasts. At intervals, the amount of luciferase mRNA was checked (to determine half-life), and the amount of luciferase activity (which reflects the amount of translation product).

Pre-mRNA Polyadenylation
Most cytoplasmic mRNAs have a polyA
tail (3 end) of 50-250 Adenylates Added post-transcriptionally by an enzyme, Poly(A) Polymerase (PAP) Turns over (recycles) in cytoplasm

Functions of the PolyA Tail

1. Promotes mRNA stability
- Deadenylation (shortening of the polyA tail) can trigger rapid degradation of the mRNA 2. Enhances translation - promotes recruitment by ribosomes - bound by a polyA-binding protein in the cytoplasm called PAB1 - synergistic stimulation with Cap!

Overview of Polyadenylation Mechanism

1.
2.

3. 4.

Transcription extends beyond mRNA end Transcript is cut at 3 end of what will become the mRNA (in green) PolyA Polymerase adds ~250 As to 3 end Extra RNA (in red) degraded

Ribosome Composition (S = sedimentation coefficient)

Ribosome Source E. coli Whole Ribosome 70S Small Subunit 30S 16S RNA 21 proteins 40S 18S RNA 33 proteins Large Subunit 50S 23S & 5S RNAs 31 proteins 60S 28S, 5.8S, &5S RNAs 49 proteins

Rat cytoplasm

80S

Eukaryotic cytoplasmic ribosomes are larger and more complex than prokaryotic ribosomes.

Structure of the E. coli Ribosome

large subunit tRNA

EF-G

small subunit

mRNA location

The cutaway view at right shows positions of tRNA (P, E sites) & mRNA (as orange beads, data from Joachim Frank lab at the Wadsworth Center)

small subunit

large subunit

Sec61 channel

path of nascent protein

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.