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DNA
Reverse Transcriptase
RNA
PROTEIN
Structure of the Genetic material, DNA, in Prokaryotes & Eukaryotes Functions : DNA Replication , in Prokaryotes & Eukaryotes Structure of the Genetic material, RNA, in Prokaryotes & Eukaryotes Functions : DNA Transcription, in Prokaryotes & Eukaryotes
B : Structure and Function of The Genetic Material: Gene Expression : Lectures 5 & 6 DNA Replication, Transcription & Translation: in Prokaryotes and Eukaryotes ( DNA Synthesis, RNA Synthesis, Protein synthesis) C. Experimental Gene Manipulation: Lectures 7 & 8 Genetic Engineering Techniques: Gene cloning: Molecular Techniques: rDNA Technology DNA, RNA and Protein Extraction/ Isolation Restriction Digestion, Purification, Cloning vehicles, Ligation, Transformation and Screening, Transfection & Selection, Clone stabilization PCR and its applications DNA Synthesis, Oligonucleotide and primers D. Application of Genetic Engineering / rDNA Techniques : Biotechnology: Lectures 9 & 10 Research, Medicine, Agriculture& GM foods.
DNA is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms and some viruses. DNA is a set of blueprints needed to construct other components of cells, such as proteins and RNA molecules.
Two long strands makes the shape of a double helix. two strands run in opposite directions to each other and are therefore anti-parallel. Chemically, DNA consists of two long polymers of simple units called nucleotides, with backbones made of base, sugars and phosphate groups.
Fig : DNA double helix
The DNA double helix is stabilized by hydrogen bonds between the bases attached to the two strands.
One major difference between DNA and RNA is the sugar, with the 2deoxyribose in DNA being replaced by the alternative pentose sugar ribose in RNA.
Ribose
Size:
The DNA chain is 22 to 26 ngstrms wide (2.2 to 2.6 nanometres), and one nucleotide unit is 3.3 (0.33 nm) long.
Thymine (T)
Sugar (deoxyribose)
Thymine (T)
Cytosine (C)
Adenine (A)
Guanine (G)
Pyrimidines
Purines
Figure 10.2B
Hydrogen bonds between bases hold the strands together: A and T, C and G
Hydrogen bond
Ribbon model
Computer model
Figure 10.3D
mRNA
mRNA
Types of mutations
NORMAL GENE mRNA Protein Met Lys Phe Gly Ala
BASE SUBSTITUTION
Met
Lys
Phe
Ser
Ala
BASE DELETION
Missing
Met
Lys
Leu
Ala
His
Homologous chromosomes
Duplication
Inversion
Reciprocal translocation
Nonhomologous chromosomes
Linking nucleotides
3
5
3
Hydrogen bonds
3
Linking nucleotides:
N-H------N
3 3 N-H------O The 3-OH of one nucleotide is linked to the 5-phosphate of the next? Whatnext nucleotide
Thymine
3
2nm
3
Adenine
3
Cytosine
3
Guanine
Base pairing
5
A T
3
C G A T
3
G/C (3 hydrogen bonds) Always pairing a purine and a pyrimidine yields a constant width
T A
3
C G
5
DNA conventions
1. DNA is a right-handed helix 2. The 5 end is to the left by convention 5 -ATCGCAATCAGCTAGGTT-3 3 -TAGCGTTAGTCGATCCAA-5 sense (forward) antisense (reverse)
5 -ATCGCAATCAGCTAGGTT- 3 3 -TAGCGTTAGTCGATCCAA- 5
DNA Structure
Some more facts:
1. Forces stabilizing DNA structure: Watson-Crick-H-bonding and base stacking (planar aromatic bases overlap geometrically and electronically energy gain) 2. Genomic DNAs are large molecules: Eschericia coli: 4.7 x 106 bp; ~ 1 mm contour length Human: 3.2 x 109 bp; ~ 1 m contour length 3. Some DNA molecules (plasmids) are circular and have no free ends: mtDNA bacterial DNA (only one circular chromosome) 4. Average gene of 1000 bp can code for average protein of about 330 amino acids 5. Percentage of non-coding DNA varies greatly among organisms Organism small virus typical virus little bacterium yeast human amphibians plants # Base pairs 4 x 103 3x 5 x 106 1 x 107 3.2 x 109 < 80 x 109 < 900 x 109 105 3000 6000 30,000? ? 23,000 - >50,000 # Genes 3 Non-coding DNA very little 200 10 - 20% > 50% 99% ? > 99% very
Reading frames
Reading frame (also open reading frame): The stretch of triplet sequence of DNA that potentially encodes a protein. The reading frame is designated by the initiation or start codon and is terminated by a stop codon. - a reading frame is not always easily recognizable - each strand of RNA/DNA has three possible starting points (position one, two, or three): Position 1 CAG AUG AGG UCA GGC AUA gln met arg ser gly ile C AGA UGA GGU CAG GCA UA arg trp gly gln ala CA GAU GAG GUC AGG CAU A asp glu val arg his
Position 2
Position 3
- mutations within an open reading frame that delete or add nucleotides can disrupt the reading frame (frameshift mutation): Wildtype CAG AUG AGG UCA GGC AUA GAG gln met arg Mutant ser gly ile glu Up to 30% of mutations causing humane disease are due to premature termination of translation (nonsense mutations or frameshift)
CAG AUG AGU CAG GCA UAG AG gln met ser gln ala
Mutations
Mutation: any heritable change in DNA Sources of mutation: Spontaneous mutations: mutations occur for unknown reasons Induced mutations: exposure to substance (mutagen) known to cause mutations, e.g. X-rays, UV light, free radicals Mutations may influence one or several base pairs
a) Nucleotide substitutions (point mutation) 1) Transitions (Pu Pu; Py Py) 2) Transversions (Pu Py) b) Insertion or deletion (indels) -
In-class exercise How many transition and transversion events are possible?
2 transitions: T C; A G 4 transversions: T A; T G C A; C G one to many bases can be involved frequently associated with repeated sequences (hot spots) lead to frameshift in protein-coding genes, except when N = 3X also caused by insertion of transposable elements into genes
Weighting of mutation events plays important role for phylogenetic analyses (model of sequence evolution)
Mutations
Mutations may influence phenotype a) Silent (or synonymous) substitution nucleotide substitution without amino acid change no effect on phenotype mostly third codon position other possible silent substitutions: changes in non-coding DNA
b) Replacement substitution - causes amino acid change - neutral: protein still functions normally - missense: protein loses some functions (e.g. sickle cell anemia: mutation in -globin) c) Sense/nonsense substitution - sense: involves a change from a termination codon to one that codes for an amino acid - nonsense: creates premature termination codon
Mutation rates = a measure of the frequency of a given mutation per generation mutation rates are usually given for specific loci (e.g. sickle cell anemia) the rate of nucleotide substitutions in humans is on the order of 1 per 100,000,000 range varies from 1 in 10,000 to 1 in 10,000,000,000 every human has about 30 new mutations involving nucleotide substitutions mutation rate is about twice as high in male as in female meiosis
Mutations
A single amino acid substitution in a protein causes sickle-cell disease
What is Genome ?
Genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the noncoding sequences of the DNA.
Size (bp)
3.2 billion 2.6 billion 137 million 100 million 97 million 12.1 million 4.6 million 1.8 million
gene number
~25,000 ~25,000 13,000 25,000 19,000 6000 3200 1700
chromosome number
46 40 8 10 12 32 1 1
helicase
DNA polymerase
DNA Replication
Since the DNA molecule is very large, there must be a way to copy it faster than just unwinding from one end to the other! This happens when the DNA molecule separates at many sites, forming thousands of replication bubbles. This allows parts of the DNA message to be replicated simultaneously in many locations. DNA polymerase adds new nucleotides , while DNA ligase joints the DNA segments together.
Origin of replication
Bubble
5 end
3 end
P
3 end 5 end
Figure 10.5B
3 5
5 3
telomeres
DNA ligase
DNA replication
DNA replication, the basis for biological inheritance, is a fundamental process occurring in all living organisms to copy their DNA. In the process of "replication" each strand of the original double-stranded DNA molecule serves as template for the reproduction of the complementary strand. Two identical DNA molecules have been produced from a single double-stranded DNA molecule.
In a cell, DNA replication begins at specific locations in the genome, called "origins". Unwinding of DNA at the origin, and synthesis of new strands, forms a replication fork. In addition to DNA polymerase, the enzyme that synthesizes the new DNA by adding nucleotides matched to the template strand, a number of other proteins are associated with the fork and assist in the initiation and continuation of DNA synthesis. Cellular proofreading that ensure near perfect fidelity for DNA replication.
DNA is synthesized by extending the 3end of the primer Hydrolysis of pyrophosphate is the driving force for DNA Synthesis
G= -7 kcal/mole
DNA polymerases are processive enzymes; thus the rate of DNA synthesis is dramatically increased (~1000 bp/sec)
The thumb helps to maintain a strong association between the DNA polymerase and its substrate
The structure of a DNA replication fork. Because both daughter DNA strands are polymerized in the 5'to-3' direction, the DNA synthesized on the lagging strand must be made initially as a series of short DNA molecules, called Okazaki fragments
A Special Nucleotide-Polymerizing Enzyme Synthesizes Short RNA Primer Molecules on the Lagging Strand
RNA primer synthesis. A schematic view of the reaction catalyzed by DNA primase, the enzyme that synthesizes the short RNA primers made on the lagging strand using DNA as a template. Unlike DNA polymerase, this enzyme can start a new polynucleotide chain by joining two or three nucleoside triphosphates together. The primase synthesizes a short polynucleotide in the 5'-to-3' direction and then stops, making the 3' end of this primer available for the DNA polymerase
Replicator sequences (origin of replication) include initiator binding sites and easily unwound DNA
FINISHING REPLICATION
Type II topoisomerases are required to separate daughter DNA molecules
The telomerase is a protein RNA complex that carries an RNA template for synthesizing a repeating, G-rich telomere DNA sequence. Only the part of the telomerase protein homologous to reverse transcriptase is shown here (green). A reverse transcriptase is a special form of polymerase enzyme that uses an RNA template to make a DNA strand; telomerase is unique in carrying its own RNA template with it at all times.
Telomerase solves the end problem by extending the 3 end of the chromosome
RNA
Ribonucleic Acid
Uracil (U)
Sugar (ribose)
Structure of RNA
Single stranded Ribose Sugar 5 carbon sugar Phosphate group Adenine, Uracil, Cytosine, Guanine
Types of RNA
Three main types Messenger RNA (mRNA) transfers DNA code to ribosomes for translation. Transfer RNA (tRNA) brings amino acids to ribosomes for protein synthesis. Ribosomal RNA (rRNA) Ribosomes are made of rRNA and protein.
Table 14.2 Types of RNA Type of RNA Messenger RNA (mRNA) Functions in Nucleus, migrates to ribosomes in cytoplasm Function Carries DNA sequence information to ribosomes Provides linkage between mRNA and amino acids; transfers amino acids to ribosomes
Cytoplasm
Cytoplasm
Double-stranded RNA
Double-stranded RNA (dsRNA) is RNA with two complementary strands, similar to the DNA found in all cells. dsRNA forms the genetic material of some viruses (double-stranded RNA viruses).
Types of RNA
Type Messenger RNA Ribosomal RNA Transfer RNA Abbr mRNA rRNA tRNA Function Codes for protein Translation Translation Distribution All organisms All organisms All organisms
in post-transcriptional modification
Small nuclear RNA Y RNA Telomerase RNA snRNA Splicing and other Eukaryotes and functions archaea RNA processing, DNA Animals replication Telomere synthesis Most eukaryotes
Regulatory RNAs
Antisense RNA aRNA Transcriptional attenuation / mRNA degradation / mRNA All organisms stabilisation / Translation block
Messenger RNA
mRNA carries information about a protein sequence to the ribosomes, the protein synthesis factories in the cell. It is coded so that every three nucleotides (a codon) correspond to one amino acid. In eukaryotic cells, once precursor mRNA (pre-mRNA) has been transcribed from DNA, it is processed to mature mRNA. This removes its intronsnon-coding sections of the premRNA.
The mRNA is then exported from the nucleus to the cytoplasm, where it is bound to ribosomes and translated into its corresponding protein form with the help of tRNA. In prokaryotic cells, which do not have nucleus and cytoplasm compartments, mRNA can bind to ribosomes while it is being transcribed from DNA.
Transfer RNA
Transfer RNA (tRNA) is a small RNA chain of about 80 nucleotides that transfers a specific amino acid to a growing polypeptide chain at the ribosomal site of protein synthesis during translation.
It has sites for amino acid attachment and an anticodon region for codon recognition that site binds to a specific sequence on the messenger RNA chain through hydrogen bonding.
Ribosomal RNA
Ribosomal RNA (rRNA) is the catalytic component of the ribosomes. Eukaryotic ribosomes contain four different rRNA molecules: 18S, 5.8S, 28S and 5S rRNA. rRNA molecules are synthesized in the nucleolus. In the cytoplasm, ribosomal RNA and protein combine to form a nucleoprotein called a ribosome. The ribosome binds mRNA and carries out protein synthesis. Several ribosomes may be attached to a single mRNA at any time. rRNA is extremely abundant and makes up 80% of the 10 mg/ml RNA found in a typical eukaryotic cytoplasm.
presence of a hydroxyl group Lacks of a hydroxyl group at at the 2' position of the ribose the 2' position of the ribose sugar. sugar. RNA is usually singlestranded DNA is usually doublestranded
Genetic Codes
Virtually all organisms share the same genetic code Second Base unity of life
U UUU UUC UUA UUG CUU CUC CUA CUG phe leu UCU UCC UCA UCG CCU CCC CCA CCG ACU ACC ACA ACG GCU GCC GCA GCG C UAU UAC UAA UAG CAU CAC CAA CAG AAU AAC AAA AAG GAU GAC GAA GAG A tyr stop stop his gln asn lys asp glu UGU UGC UGA UGG CGU CGC CGA CGG AGU AGC AGA AGG GGU GGC GGA GGG G cys stop trp arg U C A G U C A G U C A G U C A G
ser
First Base
leu
pro
Third B
AUU AUC ile AUA AUGmet (start) GUU GUC GUA GUG val
ser arg
thr
ala
gly
Stop UAA UAG UGA Arginine CGU CGC CGA CGG Lysine AAA AAG
Stop UGA
Tryptophan UGG
Leucine CUU CUC CUA CUG Isoleucine AUU AUC AUA Valine GUU GUC GUA GUG
Methionine AUG
Concept Map
RNA can be
Messenger RNA
Ribosomal RNA
Transfer RNA
also called
which functions to
also called
which functions to
also called
mRNA
Carry instructions
rRNA
tRNA
from
to
to make up
DNA
Ribosome
Ribosomes
RNA structure
RNA ribonucleic acid 3 major types of RNA
messenger RNA (mRNA); template for protein synthesis transfer RNA (tRNA); adaptor molecules that decode the genetic code ribosomal RNA (rRNA); catalyzing the synthesis of proteins
Pyrimidine (C4N2H4)
Purine (C5N4H4)
+ phosphate
--
OH
5 CH2 4
O
1
O H
H
3 OH
H
2 OH
sugar
RNA base composition: A+G/ U+C = Chargaffs rule does not apply (RNA usually prevails as single strand)
RNA structure: - usually single stranded - many self-complementary regions RNA commonly exhibits an intricate secondary structure (relatively short, double helical segments alternated with single stranded regions) - complex tertiary interactions fold the RNA in its final three dimensional form - the folded RNA molecule is stabilized by interactions (e.g. hydrogen bonds and base stacking)
RNA structure
Primary structure
A) single stranded regions formed by unpaired nucleotides
Secondary structure C
B) duplex double helical RNA (A-form with 11 bp per turn) C) hairpin duplex bridged by a loop of unpaired nucleotides D) internal loop
D E F B A G
nucleotides not forming Watson-Crick base pairs E) bulge loop unpaired nucleotides in one strand, other strand has contiguous base pairing F) junction three or more duplexes separated by single stranded regions G) pseudoknot tertiary interaction between bases of hairpin loop and outside bases
RNA structure
Primary structure
Secondary structure C
Tertiary structure
D E F B A G
RNA structure
How to predict RNA secondary/tertiary structure?
Probing RNA structure experimentally: - physical methods (single crystal X-ray diffraction, electron microscopy) - chemical and enzymatic methods - mutational analysis (introduction of specific mutations to test change in some function or protein-RNA interaction) Thermodynamic prediction of RNA structure: - RNA molecules comply to the laws of thermodynamics, therefore it should be possible to deduce RNA structure from its sequence by finding the conformation with the lowest free energy - Pros: only one sequence required; no difficult experiments; does not rely on alignments - Cons: thermodynamic data experimentally determined, but not always accurate; possible interactions of RNA with solvent, ions, and proteins Comparative determination of RNA structure: - basic assumption: secondary structure of a functional RNA will be conserved in the evolution of the molecule (at least more conserved than the primary structure); when a set of homologous sequences has a certain structure in common, this structure can be deduced by comparing the structures possible from their sequences - Pros: very powerful in finding secondary structure, relatively easy to use, only sequences required, not affected by interactions of the RNA and other molecules - Cons: large number of sequences to study preferred, structure constrains in fully conserved regions cannot be inferred, extremely variable regions cause problems with alignment
DNA carries information that can be used to construct the proteins which form structures and regulate the bodys activities. Protein synthesis involves two processes: transcription and translation. In transcription the DNA message is converted into an RNA molecule. In translation the RNA message is used to assemble amino acids into a protein chain.
mRNA
Carries instructions from DNA to the rest of the ribosome. Tells the ribosome what kind of protein to make Acts like an email from the principal to the cafeteria lady.
codon 3
serine
codon 4
isoleucine
codon 5
glycine
codon 6
alanine
codon 7
stop codon
rRNA
Part of the structure of a ribosome Helps in protein production
tRNA
A go-getter. Gets the right parts to make the right protein according to mRNA instructions
RNA Processing
Introns are pulled out and exons come together. End product is a mature RNA molecule that leaves the nucleus to the cytoplasm. Introns bad Exons good!
RNA Processing
pre-RNA molecule exon intron exon intron exon
intron
exon
splicesome
intron
exon
splicesome
exon
exon
exon
exon
Transcription
RNA molecules are produced by copying part of the nucleotide sequence of DNA into complementary sequence in RNA, a process called transcription. During transcription, RNA polymerase binds to DNA and separates the DNA strands. RNA polymerase then uses one strand of DNA as a template from which nucleotides are assembled into a strand of mRNA.
TRANSCRIPTION
RNA
TRANSLATION
Protein
In prokaryotes, RNA polymerase binds to the -10 and 35 regions of the promoter relative to the start site of transcription (+1)
promoter
operator
Reverse transcription
Reverse transcribing viruses replicate their genomes by reverse transcribing DNA copies from their RNA; These DNA copies are then transcribed to new RNA. Retrotransposans also spread by copying DNA and RNA from one another.
Transcription
Transcription, is the process of creating an equivalent RNA copy of a sequence of DNA. Transcription is the first step leading to gene expression. DNA
transcription
RNA.
reverse transcription
During transcription, a DNA sequence is read by RNA polymerase, which produces a complementary, antiparallel RNA strand. Transcription results in an RNA complement that includes uracil (U) instead of thymine (T).
Transcription process
The stretch of DNA transcribed into an RNA molecule is called a transcription unit and encodes at least one gene. If the gene transcribed encodes for a protein, the result of transcription is messenger RNA (mRNA). This mRNA will be used to create that protein via the process of translation. Alternatively, the transcribed gene may encode for either rRNA or tRNA, other components of the protein-assembly process, or other ribozymes. A DNA transcription unit encoding for protein (the coding sequence) and regulatory sequences that direct and regulate the synthesis of that protein.
DNA is read from 3' 5' during transcription. the complementary RNA is created from the 5' 3' direction. only one of the two DNA strands, called the template strand, is used for transcription because RNA is only single-stranded. The other DNA strand is called the coding strand.
RNA polymerase
DNA of gene
Terminator DNA
Elongation
Introns removed
Tail
CYTOPLASM
The DNA gene is first copied and edited into a transcript made of RNA, employing similar nucleic acid bases, except that DNAs thymine is replaced by uracil. This messenger RNA (mRNA) version of the gene is then read by cellular machinery, three letters at a time, while tiny cellular butlers known as transfer RNAs (tRNA) fetch the specified amino acids to be strung together.
Anti-Codon
Messenger RNA
RIBOSOME
There are hundreds of possible Amino Acids but these 20 Amino Acids are used by life on earth.
Amino Acid Name Glycine Alanine Valine Leucine Isoleucine Serine Threonine Cysteine Methionine Glutamic Acid Aspartic Acid Lysine Arginine Asparagine Glutamine Phenylalanine Tyrosine Tryptophan Proline Terminator
Triplet Code or Codon GGT,GGC,GGA,GGG GCT,GCC,GCA,GCG GTT,GTC,GTA,GTG TTG,TTA,CTT,CTC,C TA,CTG ATT,ATC,ATA TCT,TCC,TCA,TCG,A GT,AGC ACT,ACC,ACA,ACG TGT,TGC ATG GAA,GAG GAT,GAC,AAT,AAC AAA,AAG CGT,CGC,CGA,CGG, AGA,AGG AAT,AAC GAA,GAG TTT,TTC TAT, TAC TGG CCT,CCC,CCA,CCG TAA,TAG,TGA
3-Letter Nickna me Gly Ala Val Leu Ileu Ser Thr Cys Met Glu Asp Lys Arg Asn Gln Phe Tyr Trp Pro End
20 types of Aminoacyl-tRNA Synthetases match 20 different amino acids to tRNAs with the correct anti-codon. tRNAs
Ribosomes
Large subunit
P Site
A Site
mRNA
A U G
Small subunit
C U A C U U C G
Pre-mRNA
Eukaryotic Cell
RNA Processing
mRNA Ribosome
Translation
Protein
Translation
Three parts: 1. initiation start codon (AUG) initiation: 2. elongation elongation: 3. termination stop codon (UAG) termination: Lets make a PROTEIN!!!! PROTEIN!!!!.
Translation
Large subunit
P Site
A Site
mRNA
A U G
Small subunit
C U A C U U C G
Initiation
aa1 aa2
hydrogen bonds
U A C A U G codon
G A U C U A C U U C G A
mRNA
Elongation
aa1
G A A
hydrogen bonds
U A C A U G codon
G A U C U A C U U C G A
mRNA
aa1
1-tRNA
U A C
(leaves) 2-tRNA
3-tRNA
G A A
A U G
G A U C U A C U U C G A
mRNA
aa1
G C U
A U G
G A U G A A C U A C U U C G A A C U
mRNA
aa1
2-tRNA 4-tRNA
G A U
(leaves) 3-tRNA
G C U
A U G
G A A C U A C U U C G A A C U
mRNA
aa1
aa5
5-tRNA
U G A
3-tRNA 4-tRNA
G A A G C U G C U A C U U C G A A C U
mRNA
aa1 aa2
aa5
aa4
5-tRNA 3-tRNA
U G A
4-tRNA
G A A
G C U G C U A C U U C G A A C U
mRNA
aa4
aa5
aa199 aa200
Termination
200-tRNA
A C U
mRNA
C A U G U U U A G
End Product
The end products of protein synthesis is a primary structure of a protein protein. A sequence of amino acid bonded together by peptide bonds bonds.
aa2 aa1 aa3 aa4 aa5
aa199
aa200
Question:
The anticodon UAC belongs to a tRNA that recognizes and binds to a particular amino acid. acid. What would be the DNA base code for this amino acid?
Answer:
tRNA mRNA DNA - UAC (anticodon) (anticodon) - AUG (codon) (codon) - TAC