Molecular Biology: DNA Structure & Function

Nucleic Acids

• Nucleic acids are biopolymers composed of nucleotides, aldopentoses (either ribose or deoxyribose), and a purine or pyrimidine base linked to a phosphate
• DNA and RNA are chemical carriers of genetic information
• A nucleotide consists of a nitrogen-containing heterocyclic base, a ribose or deoxyribose sugar ring, and a phosphoric acid unit

Structure of the Nucleotide

• DNA and RNA are polymers
• The monomer units are called nucleotides
• Nucleotides are made up of a nitrogen containing heterocyclic base, a pentose (five-carbon) sugar and a phosphate group

DNA and RNA

• Deoxyribonucleic acid (DNA) and Ribonucleic acid (RNA) are the chemical carriers of genetic information
• Nucleic acids are biopolymers made of nucleotides and aldopentoses linked to a purine or pyrimidine and a phosphate

Sugars in DNA and RNA

• RNA is derived from ribose
• DNA is derived from 2'-deoxyribose
• The prime symbol (') is used to refer to positions on the sugar portion of a nucleotide

Major Purine Bases

• Adenine (A)
• Guanine (G)
• These are found in both DNA and RNA

Major Pyrimidine Bases

• Cytosine (C)
• Thymine (T)
• Uracil (U)
• Cytosine is found in both DNA and RNA
• Thymine is found in DNA
• Uracil is found in RNA

Nucleotides

• Nucleotides are the repeating units of DNA or RNA polymers
• In DNA and RNA, the base is bonded to the 1' carbon of the sugar; the phosphate bonds to the 5' carbon (connected to the 3' of the next unit)

Naming Nucleotides

• Begin with the base name
• Remove -ine ending and replace with -osine (for pyrimidines) or -idine (for purines). Uracil: replace -acil with -idine
• Add ribose or deoxyribose to denote the sugar
• Add prefix based on the number of phosphoryl groups (e.g., mono-, di-, triphosphate)

The Deoxyribonucleotides

• 2'-Deoxyadenosine 5'-phosphate
• 2'-Deoxythymidine 5'-phosphate
• 2'-Deoxyguanosine 5'-phosphate
• 2'-Deoxycytidine 5'-phosphate

The Ribonucleotides

• Adenosine 5'-phosphate
• Guanosine 5'-phosphate
• Cytidine 5'-phosphate
• Uridine 5'-phosphate

Generalized Structure of DNA

• DNA is a double helix with a sugar-phosphate backbone on the outside and base pairs in the middle
• Nucleotides are linked in a 5' to 3' direction

DNA-Secondary Structure

• The most common form of DNA is the B form
• The DNA structure was determined by Watson and Crick in 1953
• This structure is a right-handed double helix
• The strands run antiparallel
• Complementary base pairs are held together by hydrogen bonds: A=T, G=C

H-bonding in DNA Structure

• Hydrogen bonding between complementary base pairs holds the two DNA strands together
• A-T base pairs have two hydrogen bonds
• G-C base pairs have three hydrogen bonds

H-Bonds in DNA

• G-C base pairs have three hydrogen bonds

A-T Base Pairing

• A-T base pairs have two hydrogen bonds

B DNA segment

• Sugar-phosphate backbone outside the helix
• Hydrogen bonded base pairs inside the core of the helix

Grooves in DNA

• Two continuous grooves (major and minor).
• The sugar-phosphate backbone runs outside the helix
• Hydrogen bonds between base pairs inside

B DNA Structure

• Outside diameter; 2 nm
• Interior diameter; 1.1 nm
• Length of one turn of a helix is 3.4 nm and contains 10 base pairs

Nucleic Acid Sequences

• Differences arise from the sequence of bases
• A nucleic acid is a polymer made of nucleotides linked together

Describing a Sequence

• A typical sequence is written from the 5' end
• Using abbreviations (A for adenosine, G for guanosine, C for cytidine, T for thymine, U for uracil in RNA)

Learning Check NA1

• Complementary base sequence for -A-G-T-C-C-A-A-T-G-C-: -T-C-A-G-G-T-T-A-C-G-

Chromosomes

• Chromosomes are pieces of DNA that contain genetic instructions (genes)
• Prokaryotes have a single, circular, supercoiled chromosome in a region called the nucleoid
• Eukaryotes have multiple linear chromosomes within a nucleus, with membrane-bound organelles

RNA Structure

• Sugar-phosphate backbone for ribonucleotides (linked by 3' to 5' phosphodiester bonds)
• RNA molecules are usually single-stranded
• Ribose replaces deoxyribose in RNA
• Uracil replaces thymine in RNA
• Base pairing between U and A, and G and C, can form double-stranded portions in RNA

Nucleic Acids and Heredity

• Processes transferring genetic information:
  • Replication: produces identical copies of DNA
  • Transcription: reads genetic messages and carries them to ribosomes for protein synthesis
  • Translation: genetic messages are decoded to make proteins

DNA Replication

• DNA replicates itself every cell division to maintain correct genetic information
• Two strands of DNA unwind, each strand acts as a template for a new strand, and new bases pair with their complementary base
• Two double helixes form, which are copies of the original DNA

DNA Unwinds

• G-C, A-T, C-G, T-A

DNA Copied With Base Pairs

• Two copies of the original DNA strand

DNA Replication (part 2: Replication Initiation)

• DNA replication is semi-conservative
• Each parental strand acts as a template for a new, complementary strand

DNA Replication (part 2: Replication Initiation)

• DNA unwinds, and hydrogen bonds between the two strands are broken

DNA Replication (part 2: Replication Initiation)

• This process is aided by the enzyme helicase
• Single-strand binding proteins (SSBs) prevent the separated strands from rejoining

DNA Replication (part 2: Replication Initiation)

• This creates a replication bubble
• Replication bubbles form at multiple sites along the DNA molecule to speed up replication

DNA Replication (part 3: Replication of the Leading Strand)

• Once strands are unwound and separated, DNA polymerase begins building a new strand

DNA Replication (part 3: Replication of the Leading Strand)

• DNA polymerase builds the new strand in a 5' to 3' direction

DNA Replication (part 3: Replication of the Leading Strand)

• DNA polymerase can't build a new strand from scratch; it can only build onto a pre-existing strand

DNA Replication (part 3: Replication of the Leading Strand)

• RNA primase synthesizes the first nucleotides of the new strand

DNA Replication (part 3: Replication of the Leading Strand)

• The RNA primer provides a free 3' end to bind to

DNA Replication (part 3: Replication of the Leading Strand)

• DNA polymerase can now assemble complementary nucleotides as it moves along the parent strand

DNA Replication (part 3: Replication of the Leading Strand)

• DNA polymerase reads the parent strand in a 3' to 5' direction while building the new strand in a 5' to 3' direction

DNA Replication (part 3: Replication of the Leading Strand)

• The helix continues to unwind and open, allowing the leading strand to grow continuously towards the replication fork

DNA Replication (part 3: Replication of the Leading Strand)

• Later a different kind of DNA polymerase replaces the RNA primer with DNA

DNA Replication (part 3: Replication of the Leading Strand)

• How the new DNA strand is formed

DNA Replication (part 4: Replication of the Lagging Strand)

• The lagging strand is built in the opposite direction that the helix unwinds

DNA Replication (part 4: Replication of the Lagging Strand)

• First, RNA primase adds a section of RNA primer

DNA Replication (part 4: Replication of the Lagging Strand)

• Next, DNA polymerase begins to synthesize the new strand of DNA

DNA Replication (part 4: Replication of the Lagging Strand)

• Before more lagging strand can be built, the helix must continue to unwind

DNA Replication (part 4: Replication of the Lagging Strand)

• Thus, the lagging strand is built discontinuously
• Discontinuous sections are called Okazaki fragments

DNA Replication (part 4: Replication of the Lagging Strand)

• As in the leading strand, a different DNA polymerase changes the RNA primer into DNA

DNA Replication (part 4: Replication of the Lagging Strand)

• Then, ligase joins the segments of DNA together

DNA Replication (part 4: Replication of the Lagging Strand)

• Replication continues in this manner along the lagging strand, building in sections as the helix unwinds

DNA Replication (part 5: Bringing It All Together)

• Now let's look at the entire replication bubble

DNA Replication (part 5: Bringing It All Together)

• Leading and lagging strands begin to replicate, working in opposite directions

DNA Replication (part 5: Bringing It All Together)

• Meanwhile, another leading strand is replicating on the other strand of the bubble

DNA Replication (part 5: Bringing It All Together)

• There is a second lagging strand at the opposite end

DNA Replication (part 5: Bringing It All Together)

• Next, a second DNA polymerase adds deoxyribose, changing the sections of RNA into DNA

DNA Replication (part 5: Bringing It All Together)

• Finally, ligase joins the loose segments of DNA together

DNA Replication (part 5: Bringing It All Together)

• Now, let's see the entire replication bubble in action

DNA Replication (part 5: Bringing It All Together)

• There are multiple replication bubbles along the DNA molecule

DNA Replication (part 5: Bringing It All Together)

• They continue to grow until they join together

DNA Replication (part 5: Bringing It All Together)

• Now there are two complete molecules of DNA

DNA Replication (part 5: Bringing It All Together)

• Each new DNA molecule will be able to replicate and start the process all over again

Summary: DNA Replication (part 1: Opening up the Superstructure)

• During replication, the very condensed superstructure of chromosomes is opened

Summary: DNA Replication (part 1: Opening up the Superstructure)

• One step of this mechanism involves acetylation and deacetylation of key lysine residues

Summary: DNA Replication (part 2: Relaxation of Higher Structures of DNA)

• Tropoisomerases help relax supercoiled DNA by introducing single- or double-strand breaks

Summary: DNA Replication (part 3: Unwinding the DNA Double Helix)

• Replication of DNA starts with unwinding

Summary: DNA Replication (part 3: Primer/Primases)

• Primers are short oligonucleotides (4-15 nucleotides long)

Summary: DNA Replication (part 4: DNA Polymerases)

• DNA polymerases are key enzymes in replication

Summary: DNA Replication (part 4: DNA Polymerases)

• In the absence of DNA polymerase, alignment is slow

Summary: DNA Replication (part 4: DNA Polymerases)

• Along the lagging strand (3' to 5'), polymerases can synthesize only short fragments

Summary: DNA Replication (part 4: DNA Polymerases)

• These short fragments are called Okazaki fragments

Summary: Mutation and Repair

• Mutations are mistakes introduced into the DNA sequence
• Mutations can be classified as point mutations, deletions, or insertions
• Mutagens cause base changes

Summary: UV Damage and DNA Repair

• UV light causes pyrimidine dimer formation on DNA strands
• Failure to repair these dimers can result in xeroderma pigmentosum, a genetic skin disorder

Summary: DNA Repair

• Cell viability depends on DNA repair enzymes

Summary: DNA Repair (Base Excision Repair)

• A DNA glycosylase recognizes the damaged base

Summary: DNA Repair (Base Excision Repair)

• It catalyzes the hydrolysis of the ẞ-N-glycosidic bond

Summary: DNA Repair (Base Excision Repair)

• The damaged base is flipped out and removed—the sugar-phosphate backbone remains intact

Summary: DNA Repair (BER cont'd)

• At the AP site, an endonuclease catalyzes hydrolysis of the backbone
• An exonuclease removes the damaged site
• DNA polymerase inserts the correct nucleotide
• DNA ligase seals the backbone

Summary: DNA Repair (NER—Nucleotide Excision Repair)

• NER removes and repairs 24-32 units through a similar mechanism involving a number of repair enzymes

The Central Dogma

• Replication, transcription, translation are the main processes in gene expression in a typical, single-celled organism

Classes of RNA Structure

• Transfer RNA (tRNA): transfers amino acids
• Ribosomal RNA (rRNA): forms ribosomes
• Messenger RNA (mRNA): directs the amino acid sequence of proteins

tRNA

• There is at least one tRNA for each amino acid

tRNA

• Intrachain hydrogen bonding creates stems and loops within the molecule, forming a cloverleaf structure

tRNA

• Amino acid attaches to the 3' end

Transcription

• Transcription is the process wherein a messenger RNA (mRNA) is synthesized along a template strand of DNA, specifically near a gene

Transcription

• Transcription happens in the nucleolus of a eukaryotic cell

Transcription (Initiation)

• The promoter signals where transcription begins

Transcription (Initiation)

• RNA polymerase locates and binds to the promoter

Transcription (Initiation)

• A group of proteins called transcription factors locate and bind to the TATA box

Transcription (Initiation)

• RNA polymerase II binds to the promoter

Transcription (Initiation)

• Aided by transcription factors, RNA polymerase II locates the start point and begins to unwind the DNA helix

Transcription (Elongation)

• RNA polymerase II assembles the RNA nucleotides that complement the template strand

Transcription (Elongation)

• RNA polymerase reads the template strand in a 3' to 5' direction, building the new RNA strand in a 5' to 3' direction

Transcription (Elongation)

• RNA contains the nitrogenous base uracil (U) instead of thymine (T)

Transcription (Elongation)

• RNA polymerase continues along the template as the DNA strands rejoin

Transcription (Termination)

• As the RNA transcript nears completion, the enzyme encounters a terminator sequence

Transcription (Termination)

• The RNA polymerase transcribes the terminator sequence and then continues about 10–15 nucleotides before releasing the pre-mRNA strand

Summary: Transcription

• Transcription starts with DNA unwinding near the gene
• Only one DNA strand is transcribed
• Ribonucleotides assemble along the strand in a sequence complementary to the template strand
• Enzymes called polymerases catalyze the process

Summary: Transcription

• Eukaryotic genes have two parts: structural genes and regulatory genes

Summary: Transcription

• Structural genes are made of exons and introns
• Regulatory genes have control elements, one of which is the promoter

Summary: Transcription

• Promoters are unique to each gene and have initiation signals

Summary: Transcription

• Promoters contain consensus sequences

Summary: Transcription

• A TATA box lies approximately 25 base pairs upstream

Summary: Transcription

• RNA polymerases interact with their promoter regions through transcription factors, which are binding proteins

Summary: Transcription

• After initiation, RNA polymerases zip up complementary bases in a process called elongation

Summary: Transcription

• Elongation is in the 5' -> 3' direction

Summary: Transcription

• Termination is present at the end of each gene

Summary: Transcription

• Poly II has two forms—unphosphorylated for initiation and phosphorylated for elongation—and is recycled between those roles

Summary: Transcription

• RNA products of transcription may not be functional RNA immediately

Summary: Transcription

• RNA products of transcription become functional through post-transcription modification

RNA Processing

• mRNA is capped at both ends, the 5' end acquires a methylated guanine, and the 3' end acquires a poly-A tail (100-200 adenines)

RNA Processing

• Introns are spliced out

RNA Processing

• tRNA is trimmed, capped, and methylated

RNA Processing

• Functional rRNA undergoes post-transcription modification

Translation

• Translation is the synthesis of a polypeptide strand using genetic information present in the mRNA molecule

Translation (Initiation)

• Initiation begins when the mRNA transcript comes together with the smaller of two ribosomal subunits and a tRNA molecule

Translation (Initiation)

• The tRNA molecule carries the first amino acid of the polypeptide

Translation (Initiation)

• The tRNA molecule attaches to the start codon (AUG)

Translation (Initiation)

• Now the larger ribosomal subunit arrives

Translation (Initiation)

• This ribosomal subunit has 3 tRNA binding sites (E, P, A)

Translation (Elongation)

• The mRNA transcript continues across the ribosome with the amino acids bonded by peptide bonds

Translation (Elongation)

• Codons are recognized: each codon codes for a particular amino acid

Translation (Elongation)

• The incoming tRNA contains an anticodon which complements the codon in the A site

Translation (Elongation)

• Hydrogen bonds hold the tRNA in place

Translation (Elongation)

• The tRNA carries the next amino acid in the chain

Translation (Elongation).

• Now the large ribosomal subunit joins the two amino acids together with a peptide bond.

Translation (Elongation)

• The tRNAs, together with the mRNA template, move down one site

Translation (Elongation)

• The used tRNA is released from the E site

Translation (Elongation)

• The entire process repeats

Translation (Termination)

• Translation is terminated when a stop codon is reached

Translation (Termination)

• The release factor is a protein that recognizes and binds to the stop codon

Translation (Termination)

• The release factor adds a water molecule, or hydrolyzes the chain, releasing the polypeptide.

Translation (Termination)

• Once the polypeptide is released, the ribosomal subunits and other factors break apart

Translation (Overview)

• Multiple ribosomes travel along the mRNA strand simultaneously translating copies of the polypeptide

Gene Regulation

• Gene regulation by various methods controls which genes are expressed and when
• Some regulations operate at the transcriptional level (DNA - > RNA)
• Other regulations operate at the translational level (mRNA - > Protein)

Gene Regulation: Transcriptional Level (Promoters)

• Promoters are located adjacent to the transcription site
• Defined by an initiator and conserved sequences such as TATA or GC boxes
• Transcription factors bind to different modules of the promoter
• Transcription factors allow the rate of mRNA synthesis to vary significantly

Gene Regulation: Transcriptional Level (Promoters)

• Transcription factors find their targeted sites by twisting their protein chains so that a certain amino acid sequence is present at the surface.

Gene Regulation: Transcriptional Level (Promoters)

• One such conformational twist is provided by metallic binding fingers (next screen).
• Two other prominent transcription factor conformations are: the helix-turn-helix and the leucine zipper.

Gene Regulation: Transcriptional Level (Promoters)

• Transcription factors also possess repressors that reduce the rate of transcription.

Gene Regulation: Transcriptional Level (Enhancers)

• Enhancers speed up transcription.

Gene Regulation: Transcriptional Level (Enhancers)

• Enhancers can be thousands of nucleotides away from the transcription site.
• DNA loops bring the enhancer to the initiation site.

Gene Regulation: Transcriptional Level (Response Elements)

• Response elements are activated by their transcription factors in response to an outside stimulus. This stimulus could be heat shock, heavy metal toxicity or hormonal signals.

Translation Level (AARS Control)

• Each amino acid must bond to the correct tRNA.

Translation Level (AARS Control)

• Enzymes called aminoacyl-tRNA synthetases (AARS) catalyze this bonding.

Translation Level (AARS Control)

• Each AARS recognizes its tRNA by specific nucleotide sequences

Translation Level (AARS Control)

• The active site of each AARS has two sieving sites

Translation Level (Termination Control)

• The stop codons must be recognized by release factors

Translation Level (Termination Control)

• The release factor combines with GTP

Translation Level (Post-translational Control)

• In most proteins, the Methionine (Met) at the N-terminal end is removed by Met-aminopeptidase.

Translation Level (Post-translational Control)

• Proteins called chaperones help newly synthesized proteins fold into their proper conformation

Translation Level (Post-translational Control)

• If rescue by chaperones fails, proteasomes may degrade the misfolded protein

Recombinant DNA

• Restriction enzymes cut the DNA backbone at specific sequences

Recombinant DNA

• Donor and plasmid DNA are cleaved by the same restriction enzyme

Recombinant DNA

• Donor DNA fragments join to complimentary plasmid fragments through hydrogen bonding

Recombinant DNA

• Plasmid ring is restored using DNA ligase

Recombinant DNA

• Engineered plasmid is introduced into a bacterium to be reproduced

Polymerase Chain Reaction (PCR)

• DNA is mixed with Taq polymerase, a primer sequence for a specific gene and nucleotide triphosphates

Polymerase Chain Reaction (PCR)

• A thermocycler raises and lowers the temperature to separate and anneal DNA strands, and to allow for DNA strand elongation

Polymerase Chain Reaction (PCR)

• Repeating the cycle doubles the new DNA strands

PCR: Heating and Reaction

• The subject DNA is heated with Taq polymerase and Mg2+ to separate strands

PCR: Heating and Reaction

• Deoxynucleotide triphosphates are also added, along with primers to aid in DNA strand elongation.

PCR: Annealing and Growing

• Temperature is reduced to 37-50 C to allow primers to hybridize to the complementary sequences

PCR: Annealing and Growing

• Primers are added to create complementary sequences

PCR: Taq Polymerase

• Taq polymerase raises temperature to 72°C to add nucleotides to the two primed DNA strands

PCR: Growing More Chains

• Repeating the denature, anneal, and synthesize cycles doubles the new DNA strands exponentially.

PCR: Growing More Chains

• PCR is now automated and can be carried out in a similar amount of time as it takes to make a cup of tea

PCR: Growing More Chains (data given)

• The table contains data for oligo sequence, primer and type. Each sequence with a given type is listed.