SCSS 2QMT

Biochemistry lec
UNIT 7: NUCLEIC ACIDS
NOTE:
 Temperature to break all H bonds: Temperature  # of G-C
Historical Perspective
Fredric Miescher - In 1968, he isolated nuclein and protamine from salmon sperm.
Oswald, Avery, Colin, Macleod & Maclyn McCarty - In 1944, they showed that DNA from pathogenic strains of bacterium
Pneumococcus could be transferred into nonpathogenic
strains, making it pathogenic. Dead bacteria+ nonvirulent
bacteria= death of mouse (due to nonvirulent bacteria  virulent)
Alfred Hershey and Martha Chase - In 1952, they proved that DNA, and protein, is the genetic material of
T2 bacteriophage. When a virus infect hosts, they share DNA
hence there is transfer of genetic info.
James Watson and Francis Crick - They proposed the model of the double helix of DNA in 1953.
Roselin Franklin - Has the data/evidence of the double helix structure.

Nucleic Acids - Biomolecules that store information for cellular growth and
reproduction.
Types of Nucleic Acids: Deoxyribonucleic Acid (DNA) - More stable due to one oxygen group.
Ribonucleic Acid (RNA) - Less stable
Nucleotides - Are long chains of monomers that make up the polymers.
Nitrogen Heterocyclic base, Pentose Sugar, Phosphate residue - Each nucleotide is made up of this.

Nucleotide Bases: Derivatives of pyrimidine/purine base

Nucleotide Bases - Are aromatic, strongly absorbs UV light and exhibits keto-enol
Tautomerism (allowing stable H bonds)
Nucleoside - Pentose sugar and nucleotide base linked by N-glycoside bond. Has
sugar and nitrogenous base without a phosphate
group.
Ribonucleoside - β-D-ribose sugar (2’-OH)
Deoxyribonucleoside - β-D-2-deoxyribose (2’-H)
Glycosidic Bond - A bond between sugar and nitrogenous base.
Purine - βC1’- N9
Pyrimidine - βC1’- N1
SCSS 2QMT
-sine - The suffix of Purines
-dine - Suffix of Pyrimidine
Nucleotide - A nucleoside with one or more phosphate groups bonded to the 3’ and
5’ position of the pentose.
Covalent bonds/ Ester Bonds -^ are bonded by these bonds.
Phosphate Ester Bonds - Bond between a sugar and a phosphate. Are covalent bonds.
ATP - An example of a nucleotide with a nitrogenous base. A purine.
GTP -^ but if triphosphate is not present.

Nucleic Acid Structure

3’-5’ Phosphodiester bonds - This link nucleotide residues in nucleic acid.
5’  3 - Direction of polynucleotide strands. How it is read.
Phosphodiester linkage - Links between phosphates and two sugars.
Sugar+ Phosphate - The backbone for DNA structure
Primary Structure - Sequence of bases along the pentose phosphodiester backbone of the
nucleic acid. Read from ‘5 to ‘3.
Single Letter (A, G, C, U, T) - System of notation is this.
A,G -The purines
T,C - The perimidines
Secondary Structure - Structure where bases are approximately perpendicular to the axis of
the helix and are stacked one top of another like a pile of
plates.
B-DNA - The physiological form. The common form of DNA.
2 antiparallel polynucleotide strands - ^ Is made up of this which are coiled together.
Right-handed helix (clockwise) - ^ forms this.
Sugar-phosphate backbone - These are negatively charges and are on the outside of the helix.
A-T and G-C -Hydrogen bonding between these base pairs holds the two
polynucleotide strands together.
SCSS 2QMT
2 H bonds - Number of H bonds in A-T
3 H bonds - Number of H bonds in G-C, making it the basis of the temp to which
can break all H bonds of a nucleic acid.
Major Groove and Minor Groove - Spaces between adjacent turns of the helix form these two grooves
3 Major Forces Contributing to Helix Formation
1. H-bonding in base pairing
2. Hydrophobic interactions in base stacking
a. Hydrophobic due to 2 planar aromatic rings.
3. Dipole-dipole interactions.
FORMS OF DNA
B-DNA - The normal form, what we usually see. Right handed helix.
Aqueous Environment -^ is found in this environment.
Right-Handed Helix -^ is in this helix formation
A-DNA - Not found in vivo. Thicker than B-DNA.
Low Humidity (dry) environments -^ is found in this environment, reason why it is thicker and clumped up
Right-Handed Helix -^ helix formation
Z-DNA - Occurs in alternating purine-pyrimidine bases
Left-handed double helix - ^ helix formation
In Vivo (but not common) -^ exists here.
Gene Expression -^ may play a role here.
Tertiary Structure - There is a further coiling and twisting of the DNA helix.
Topoisomerase - Is capable of cutting and rejoining DNA ends to produce supercoils.
Negative/Positive Supercoils - This depends on the initial structure
DNA Gyrase - This rejoins the cut DNA and then releases it for further coiling.
Prokaryotic DNA - This is circular
Eukaryotic DNA - This is linear
Quaternary Structure - This has topological changes induced by supercoiling accommodated
by histone-protein component of chromatin.
Eukaryotic DNA - This is long and linear. This is packed in a tight structure in the nucleus
2m -^ is this long
Chromatin - DNA molecules that wound around particles of histones in a beadlike
structure.
Histone - A protein where in the DNA wraps around in.
Lys and Arg -^ is rich in these two amino acids.
Eukaryotic DNA -^ is associated with this type of DNA.
How Chromosomes form:
DNA strands  Histone+DNA= Nucleosome  Chromatin  Chromatin loops  Condensed chromatin loops  Chromosome

DENATURATION AND RENATURATION OF DNA -This is transfer of information occurs from one DNA strand to another
Denaturation - Needed for replication When the DNA duplex is subjected to
conditions of pH, temperature or ionic strength that disrupts H
bonds. Hence the strands are separated.
Melting Temperature (Tm) - A measure of the base composition of DNA. Increases linearly with
the proportion of G-C bonds.
G-C bonds -^ is based from the presence of these bonds.
260nm - As strands separate, the absorbance at this measure increases.
Double Helix - This unwinds when DNA is denatured.
Renaturation - Forming of DNA strand
Cooling and annealing -^ can be done via this.

RNA: Streucture and Function

RNA - Are single stranded, classified according to structure and function.
Transfer RNA - Transports amino acids to site of protein synthesis.
Ribosomal RNA - Varies in size, combines with proteins to form ribosomes.
Ribosomes - Sites of protein synthesis.
Messenger RNA - Has variable sizes, directs amino acid sequence of proteins.
Small Nuclear RNA - Needed to initialize DNA translation. Process initial mRNA to its
mature form in eukaryotes.
Small Interfering RNA - Artificial/synthesized. Needed to study gene expression. Affects gene
expression. Used by scientists to knock out a gene being studied.
Micro RNA - More natural than siRNA, affects gene expression.
SCSS 2QMT
Growth and development -^ is important in this.
tRNA (transfer RNA) - The smallest of the three RNAs. Single stranded polynucleotide chain
between 73-94 nucleotide residues.
Amino Acid -^ carries this at its 3’ end
Intramolecular Hydrogen Bonding - ^ this bonding occurs.
rRNA (Ribosomal RNA) - Contains all the subunits for translation.
Ribosomes -^ are found here. Made up of two subunits
Ribosomes - Makes up 60-65% of the ribosome
Protein - The remaining35-40% is made up of this.
Analytical ultracentrifugation - ^ analyzed via this
Sedimentation Coefficients/ Svedberg units (S) - ^ is characterized via this
mRNA (Messenger RNA) - A ribonucleic acid that carries coded genetic information from DNA
to ribosomes for the synthesis of proteins. Short lived and
present in small amounts. Single stranded.
Biosynthesis - This is directed by information encoded on the DNA.
Unwound DNA - A complementary strand of mRNA is synthesized along this
3’ end -^ starts from this end

Unit 8: Biosynthesis of Nucleic acids (Replication)

NOTES:
 DNA replication= separation of stands + synthesis of two daughter strands complementary to parent template
 Challenges in duplication of circular double-stranded DNA
o Continuous unwinding and separation of the two DNA strands
o Protection of unwound portions from attack by nucleases that attack single-stranded DNA
o Synthesis of the SNA template from one 5’ 3’ strand and one 3’  5’ strand
o Efficient protection from errors in replication
 General Features of DNA Replication
o Each strand can serve as a template
o Semi Conservative
o Requires Primers
o Semi-discontinuous
o Usually bidirectional
o Ordered, sequential and highly accurate

Rudolf Virchow, 1858 - Where cell arises, there must be a previous cell, just as animals can
only arise from animals and plants from plants.
Copy and pass on genetic information - The most fundamental task of the cell, needed for next generation cell
DNA Replication - Process of duplicating cells T
Central Dogma - Mechanism by which information is transferred in the cell is based on this.
DNA Replication - Replication of itself
Transcription - Done to make more proteins, conversion of DNA to RNA
Reverse transcription - Conversion of RNA to DNA
RNA Replication - Occurs in the nucleus
Translation - From genetic information (RNA) to usable proteins
DNA polymerase - A protein that binds new strands to old strands
Okazaki Fragment - The template. Fixed fragment serves as a guide for the next binding.
Lagging strand -^ is connected to this strand.
Leading strand - Receives one RNA primer and is continuously extended from the
primer by DNA polymerase with high processivity.
Lagging Strand - Receives several RNA primers, extends discontinuously from each
primer forming Okazaki fragments.
SCSS 2QMT

Manners of Replication
Semi-Conservative Model - Half of the original strand is conserved and half is new. When
parental strands separate, each strand serves as template and
makes a new complementary strand.
Fully Conservative Model - After the replication of the original strand, the original strand winds
up back together.
Non-Conservative Model - After replication, what remains are all new strands.
Dispersive Model - Not seen in vivo, seen in experimentation, parent strand combines
with new strands.

Primers - Tells DNA when it is time to replicate. Directs where the DNA helix
will split.
DNA-directed DNA Polymerase - Responsible for synthesizing new DNA strands from a DNA
template.
Primer -^ requires this. This provides the 3’ -OH terminus on which to add
new nucleotides.
Polymerization - Process of forming new strands
5’-3’ Direction -^ occurs in this direction to conserve integrity of sequence.
4 DNTPs, Mg2+, RNA Primer - DNA polymerase has the following requirements.
dTTP, dATP, dGTP, dCTP - The four deoxy nucleic trisphosphates. Needed to know which base
pair to fill up.
RNA Primer - A short strand of RNA to which the growing polynucleotide chain is
covalently bonded in the early stages of replication. Helps
DNA to replicate.
Mg2+ - Phosphates are negatively charged hence this is needed to stabilize the
helix as it is being unwounded and moved in the sequence.
Deoxy Nucleic Triphosphate - DNTP stands for?
E.coli - There are at least 5 types of DNA polymerase (pol) in this amoeba.
Polymerization 5’ 3’ and Exonuclease 3’  5’ - All three Pols (Pol I, Pol II, Pol III) have this.
Exonuclease 5’  3’ - Only seen in Pol I, used to cut and repair sequencing.
5’-3’ Direction - Both the lagging and leading strand are replicated in this direction.
Leading strand - This is synthesized continuously
5’  3’ towards the replication fork -^ replicates in this direction
Lagging Strand - Synthesized discontinuously
5’  3’ away from the replication fork -^ replicates in this direction.
Origin of Replication - DNA double helix unwinds at this specific point. Its sequence will
match the RNA primer.
RNA Primer - Where replication will start
Polymerase - Splits DNA into two
Bidirectionally - Replication proceeds in this direction.
Replication Forks - In each origin of replication, this is present. Points at which new
polynucleotide chains are formed.
DnaA - This binding results to “melting” of the double helix at the origin or
replication.
Helicase - In the formation of replication forks, this is a helix-destabilizing
protein that promotes unwinding by binding at the replication fork.
Single-Stranded Binding (SSBs) proteins - This protein stabilize single-stranded regions by binding tightly to them.
SCSS 2QMT

Formation of Primosome
Primosome - The copying machine, processivity moves along the lagging strand template.
Primase, DNA Helicase, SSB Proteins -^ is made up of these components.
Primase - Enzyme that will help form the complimentary strand.
Lagging strand -^ is specific to this strand
Helicase - Promotes unwinding by binding at the replication fork.
SSB Proteins - Stabilize the strands.
Unwinding Problem
Unwinding process - Tension is built up as DNA begins with this process.
Positively supercoiled - Through ^, the DNA becomes this.
DNA Gyrase (Topoisomerase II) - This relives the tension in supercoils. Changing the DNA into
negatively supercoiled DNA.
ATP Hydrolysis -^ uses this to accomplish its task
Formation of Replisome
Replisome - A functional unit, helps link the new DNA strands together, binding
of new to original strands.
DNA Pol III, Helicase, SSBs, Primase -^ is a functional unit made up of these components.
DNA Polymerase III - The synthesis and linking of new DNA strands begins with this.
3’ -OH of RNA Primer - The newly formed DNA is linked to this.
Two tethered polymerases - This can replicate both strands by looping the DNA of the lagging
strand template back on itself, causing the template to have the
same orientation as the leading strand template.
Okazaki Fragment - Once the polymerase assembling the lagging strand reaches the 5’
end of this fragment, the lagging strand template is released,
and the polymerase begins work at the 3’ end of the next RNA
primer toward the fork (trombone model)
Removal of RNA Primase
DNA Polymerase I - Excision of RNA primers if via this. Repairs with complimentary fragment.
5’-3’ Exonuclease Activity - ^ Has this. Plays a key role in removing the RNA primer.
10 nucleotides - ^ function is to remove this from the 5’ end of a single strand nick.
DNA Ligase - In charge of nick ligation. “Cementing of strands”, fills up the gaps
made by DNA polymerase I.
Proofreading
DNA Polymerase I and III - Proofreading is done by these.
10^3 nucleotides per second - Replication takes this long.
Mutations - Errors in replication result to this.
10^9 to 10^10 - ^ occurs spontaneously only once every _____ base pairs
3’  5’ Exonuclease Activity - This removes mispaired nucleotides from the 3’ end of the growing
DNA. Key in accuracy of DNA synthesis.
DNA Pol I and III -^ is found in these polymerases
Termination of Replication
Tus Protein - Signals the stop of replication. When this binds to DNA, this signals
the primosomes to stop replication.
Termination Sequences - The orientation of ^ relies on the orientation of this.
Replication Fork -^ allows this to pass if it is moving in one direction, block progress if
this moves in the opposite direction around the genome.

Unit 9: Transcription of the genetic code

(Biosynthesis of RNA)
Transcription - Biosynthesis of RNA using DNA template by a DNA-dependent
RNA polymerase.
Nucleus - In eukaryotes, ^ happens here.
DNA template - This exists in a complementary manner.
Coding Strand - Used in transcribing RNA, goal is RNA transcript identical to
template.
RNA Polymerase - Composed of subunits, doesn’t have DNTP. Is bacterial DNA
dependent. The start of RNA transcription.
SCSS 2QMT
5’-3’ direction -^ synthesizes RNA in this direction.
ATP, GTP, CTP, UTP - All these four NTPs are required.
Mg2+ - This is also a required as a cofactor
Primer - This is not needed in RNA synthesis
α.2, β, β’, ω - the RNA polymerase is made these five core subunits. About 400kDa.
Sigma (σ) Factor - The sixth subunit, has an important role in the recognition of the
promoter. Directs the core complex to specific binding sites on
the DNA.
Promoter - DNA sequence that provide direction for RNA polymerase.

Different DNA Strands and their Role

Template (antisense) strand - Serves as the template for RNA synthesis
RNA polymerase - This binds to and transcribes only on ^.
Coding (nontemplate/sense) strand - Identical to the RNA transcribed from the gene, with the U in the
RNA in place of the T in DNA.
RNA Transcript - Identical to template, complementary to coding. Meaning it has the
same content but in antiparallel configuration.
Promoter Locus in Prokaryotic Gene - Are sequences present in DNA. What sigma factor tries to find.
Pribnow Box/ -10 region - Is 10 nucleotides bases away from starting point
-35 region - This is 35 nucleotide bases away from the starting point
UP element - Like the -35 region but more specific for bacteria. Around 40-60
nucleotide bases away.
Is this applicable to eukaryotes? Yes
Tata Box - The eukaryotic version of the prokaryotic regions. Around 50
nucleotide bases away from the starting point. Much further
promoter region.
Three Phases of Transcription
Initiation - This is dependent of the sigma factor, RNA polymerase binding to DNA.
Closed promoter complex - Recognition of a promoter region by a sigma factor
Open promoter complex - Unwinding of the DNA
-10 region - Where the opening of the helix happens.
Elongation - Technically making the RNA longer.
Purine Ribonucleotide Triphosphate - Addition of this, first base of RNA transcript. GTP to ATP.
RNA polymerase - This initiates mRNA synthesis
Sigma Factor - This dissociates as elongation proceeds
RNA polymerase around axis of DNA duplex - At this situation, there would be no strain and no supercoiling of DNA
but RNA chain would be wrapped around. Unlikely to happen
doe it would be difficult to disentangle RNA from DNA
complex. Helps stabilize DNA.
10 Base pairs - RNA wraps around the double helix every ___.
RNA elongates away from DNA duplex - When this happens, there is relaxing of positive and negative
supercoils. Usually what happens in RNA transcription.
Topoisomerase -^ happens when this is present. Could remove supercoils.
Termination - The stopping of RNA transcription.
Extrinsic Termination - The use of the rho factor, is a rho-dependent mechanism.
Rho (ρ) Factor - This recognizes the termination region. Weakens the interaction
between the template and the transcript. Slips to transcription
via mRNA.
Intrinsic Termination - The rho-independent mechanism, dependent instead of the DNA
sequence. Controlled by specific sequences, termination sites.
Palindromes -^ contains this in the template strand.
Hairpin Loop - Newly synthesized RNA form this. Where the inverted repeats would
bind together.
Dumbed down version of Transcription
1. Initiation
a. The promoter region is recognized by the sigma factor
b. RNA polymerase binds to the promoter regions (-10, -35)
SCSS 2QMT
c. Unwinding of DNA for transcription
i. Opening of helix in the -10 box
2. Elongation
a. Purine NTP adds, serves as base for RNA transcription
b. RNA polymerase initiates mRNA synthesis
i. As RNA gets longer, nawawala sigma factor
3. Termination
a. May be extrinsic or intrinsic termination

Prokaryotic Transcription
Prokaryotes - Has no nucleus.
Along DNA - RNA transcription is in this direction.
mRNA - These are synthesized on the bacterial nucleoid in direct contact and
are immediately for translation.
Eukaryotic Transcription: RNA Polymerases
RNA Polymerase I - Compartment in nucleolus
RNA Polymerase II - Compartment in nucleoplasm
RNA Polymerase III - Compartment in nucleoplasm

Processing of Eukaryotic mRNAs: Post- Transcriptional Modifications

Addition of 5’ Capping - Serves as a helmet, prevents 5’ from being digested, aids in the
transport of mRNA out of the nucleus, role in initiation of
mRNA translation.
GMP (Guanine Base with 1 Phosphate) - 5’ Cap is made up of this.
Guanylyl transferase - This adds GMP in an inverted orientation
Guanine methyltransferase - Methylation of C7 at G by this.
Splicing of Introns - When introns are transcribed, they are cut off. Eukaryote genes
frequently contain intervening base sequences that do not
appear in the final mRNA of that gene product.
Exons - Are expressed DNA sequences
Introns - Are intervening DNA sequences that are not expressed
Intron and Exons - Are transcribed, as it transcribed some areas are strictly “pre mRNA”
and are cut out, the remaining sequences are then joined
together.
Introns - Ones that are cut out
Exons - Ones that remain and are joined together.
Splicing Reaction - This is the forming of a loop within itself. Exons are separated by
intervening intron.
Lariat - When exons are spliced together, this is formed in the intron. The loop
Spliceosome-Mediated Intron Splicing - Another way of intron splicing with the use of an
additional mechanism.
Spliceosome - Made of single nucleotide RNAs. Binding of introns and pull introns
together forms this, this will remove introns from mRNA sequence.
snRNAs -^ is made up of this, associated with snRNPs.
Polyadenylation at 3’ Tail - This tail protects the mRNA from nucleases and phosphatases
Polyadenylate (Poly-A) tail - A very long chain of adenine bases.
100-200 nucleotides long -^’s length
Adenine -^ is purely made up of this.
3’ End -^ is added to this before mRNA leaves the nucleus.
SCSS 2QMT