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Transcript
Lecture 21—BCH 4053—Summer 2000
Slide 1
Nucleotide Functions
• Building blocks of nucleic acids
• Triphosphates are energy intermediates
• ATP major energy currency
• GTP involved in driving protein synthesis
• “Carriers” of metabolic intermediates
• UDP intermediates in sugar metabolism
• CDP intermediates in lipid metabolism
• NAD and CoA are ADP intermediates
• Chemical signaling “second messengers”
• cyclic AMP and cyclic GMP
Slide 2
Nucleic Acid Structure
• Linear polymer of nucleotides
• Phosphodiester linkage between 3’ and 5’
positions
• See Figure 11.17
Slide 3
Nucleic Acid Sequence
Abbreviations
• Sequence normally written in 5’-3’
direction, for example:
Guanine
Adenine
Cytosine
H
N
O
H2N
H 2N
N
N
N
N
N
N
H
H
O
H
O
H
O
O
O
O
O-
O
H
H
H
O
O
P
P
P
O-
H
H
H O
O
H O
P
O
H
H
H
H
H
O
H
H
H
H
O
O
N
N
O
H
N
O
N
O
Thymine
NH2
N
O-
O
O-
Lecture 21, page 1
Slide 4
Sequence Abbreviations, con’t.
Let Le tter sta nd for base:
A
G
C
T
5'-end
P
3'e nd
P
P
P
P
Let Letter stand for nucleoside
5'-end
pApGpCpTp
3'-end
Let Letter stand for nucleotide
5'-end
AGCT
3'-end
Slide 5
Biological Roles of Nucleic
Acids
• DNA carries genetic information
• 1 copy (haploid) or 2 copies (diploid) per cell
• See “History of Search for Genetic Material”
• RNA at least four types and functions
•
•
•
•
messenger RNA—structural gene information
transfer RNA—translation “dictionary”
ribosomal RNA—translation “factory”
small nuclear RNA—RNA processing
Slide 6
DNA Structure
• Watson-Crick Double Helix
• Clues from Chargaff’s Rules
• A=T, C=G, purines=pyrimidines
• Helical dimensions from Franklin and Wilkins
X-ray diffraction studies
• Recognition of complementary base pairing
possibility given correct tautomeric structure
(See Figure 11.20)
Lecture 21, page 2
Slide 7
Nature of DNA Helix
• Antiparallel strands
• Ribose phosphate chain on outside
• Bases stacked in middle like stairs in a spiral
staircase
• Figure 11.19—schematic representation
• Complementary strands provide possible
mechanism for replication
• Figure 11.12 representation of replication process
Slide 8
Size of DNA Molecules
• 2 nm diameter, about 0.35 nm per base pair in
length
• Very long, millions of base pairs
Organism
• SV 40 virus
• λ phage
• E. coli
• Yeast
• Human
Base Pairs
MW
Length
5.1 Kb
48 Kb
3.4x10 6
32 x 106
1.7 µm
17 µm
4,600 Kb
13,500 Kb
2.9 x 106 Kb
2.7 x 109
9 x 109
1.9 x 1012
1.6 mm
4.6 mm
0.99 m
Slide 9
Packaging of DNA
• Very compact and folded
• E. coli DNA is 1.6 mm long, but the E. coli cell
is only 0.002 mm long
Histones are rich in the basic amino
acids lysine and arginine, which
have positive charges. These
positively charged residues provide
binding for the negatively charged
ribose-phosphate chain of DNA.
• See Figure 11.22
• Eukaryotic cells have DNA packaged in
chromosomes, with DNA wrapped around an
octameric complex of histone proteins
• See Figure 11.23
Lecture 21, page 3
Slide
10
Messenger RNA
• “Transcription” product of DNA
• Carries sequence information for proteins
• Prokaryote mRNA may code for multiple
proteins
• Eukaryote mRNA codes for single protein, but
code (“exon”) might be separated by noncoding sequence (“introns”)
• See Figure 11.24
Slide
11
Ribosomal RNA
• “Scaffold” for proteins involved in protein
synthesis
• RNA has catalytic activity as the “peptidyl
transferase” which forms the peptide bond
• Prokaryotes and Eukaryotes have slightly different
ribosomal structures (See Figure 11.25)
• Ribosomal RNA contains some modified
nucleosides (See Figure 11.26)
Remember that the sedimentation
rate is related to molecular weight,
but is not directly proportional to it
because it depends both on
molecular weight (which influences
the sedimentation force) and the
shape of the molecule (which
influences the frictional force).
Slide
12
Transfer RNA
• Small molecules—73-94 residues
• Carries an amino acid for protein synthesis
• One or more t-RNA’s for each amino acid
• “Anti-codon” in t-RNA recognizes the nucleotide
“code word” in m-RNA
• 3’-Terminal sequence always CCA
• Amino acid attached to 2’ or 3’ of 3’-terminal A
• Many modified bases (Also Figure 11.26)
Lecture 21, page 4
Slide
13
Small Nuclear RNA’s
• Found in Eukaryotic cells, principally in the
nucleus
• Similar in size to t-RNA
• Complexed with proteins in small nuclear
ribonucleoprotein particles or snRNPs
• Involved in processing Eukaryotic
transcripts into m-RNA
Slide
14
Chemical Differences Between
DNA and RNA
• Base Hydrolysis
• DNA stable to base hydrolysis
• RNA hydrolyzed by base because of the 2’-OH
group. Mixture of 2’ and 3’ nucleotides
produced
• See Figure 11.29
• DNA more susceptible to mild (1 N) acid
• Hydrolyzes purine glycosidic bond, forming
apurinic acid
Slide
15
Enzymatic Hydrolysis of Nucleic
Acids
• Many different kinds of nucleases in nature
• Hydrolysis of phosphodiester bond
• Exonucleases hydrolyze terminal
nucleotides
• Endonucleases hydrolyze in middle of
chain. Some have specificity as to the base
at which hydrolysis occurs
Lecture 21, page 5
Slide
16
Enzymatic Hydrolysis, con’t.
• Specificity as to the bond which is cleaved
• a type cleaves the 3’ phosphate bond
• Produces 5’-phosphate products
• b type cleaves the 5’ phosphate bond
• See Figure 11.30
• Examples: (Also see Table 11.4)
• Snake venom phosphodiesterase
• “a” specific exonuclease
• Spleen phosphodiesterase
• “b’ specific exonuclease
• See Figure 11.31
Slide
17
Restriction Endonucleases
• Enzymes of bacteria that hydrolyze
“foreign” DNA
• Name based on “restricted growth” of
bacterial viruses
• Enzymes specific for a short sequence of
nucleotides (4-8 bases in length)
• Methylation of the same sequence protects
“self” DNA from hydrolysis
Lecture 21, page 6