SB

BSCI170 exam 4

Nucleic acids

  • All cells have DNA as their genetic material 

  • DNA is copied into RNA (A U C G) attached to a ribose sugar, which carries instructions to the ribosomes to make proteins 

    • Some RNA is used as structural elements 

Structure of DNA

  • Nucleotides 

    • Phosphate - sugar - nitrogen base 

    • Different bases (A T C G) attached to a deoxyribose sugar 

    • Types of bases 

      • Pyrimidines (single ring structure)

      • Purines (double-ring structure) 

  • Polynucleotides (polymers of nucleotide monomers), also known as nucleic acids 

    • Sugar - phosphate backbone and nitrogenous bases 

      • Phosphodiester bond 

  • Watson & Crick model 

    • Double helix 

      • Backbone on the outside 

      • Bases on the inside 

      • Strict base pairing 

        • A:T or G:C (purine : pyrimidine)

        • Constant diameter 

      • Anti-parallel strands

        • Polynucleotides run in opposite directions

          • 3’ hydroxyl 

          • 5’ phosphate 

        • Held together by hydrogen bonding (between complementary bases) 

          • Can only interact if one base is oriented upside down relative to the other 

DNA replication

  • Semi-conservative replication

    • One parent strand + one daughter strand 

    • New double helix - a hybrid of old and new 

  • Step 1: Unwind double helix 

    • Helicase enzyme 

      • Breaks hydrogen bonds 

    • Single-stranded binding protein 

      • Covers the bases so they don’t simply hydrogen bond back together 

    • Topoisomerase 

      • Second enzyme 

      • Assists helicase in unwinding the double helix

  • Step 2: Make new strands 

    • Primer 

      • Start the process of making a new daughter strand made by the enzyme primase 

    • DNA polymerase III

      • Polymer building enzyme that assembles nucleotide monomers

    • DNA polymerase I 

      • The primer-removing enzyme is needed because primers are made of RNA and not DNA 

    • Ligase 

      • DNA replication repeats along the length of the strand, making fragments that are joined together by the enzyme ligase 

Unwinding DNA

  • The replication bubble 

    • Origin(s) of replication (ori)

      • Special sequence; “where to start” 

      • Prokaryotes = one origin, small circular chromosome

      • Eukaryotes = many origins, large linear chromosomes 

    • Origin recognition proteins 

      • Bind to ori forming the origin replication complex 

        • Pulls apart the to strands of the double helix

        • Initiates a replication bubble 

    • Once small ubble is open, replication proteins can get to work 

    • Unwinding proteins 

      • + Helicase (enter e/a fork, moving awak from the origin and separating the strands, expanding the bubble 

      • + Topoisomerase (relieves the strain formed when helicase unwinds the double helix) 

      • + Single stand binding proteins (coat a single stranded DNA to prevent the two strands from coming back together) 

      • Make bubble bigger over time 

  • The replication fork 

    • Each end of the bubble isa fork 


Copying DNA strands

  • Daughter strand synthesis 

    • Same time as bubble expands 

    • Step 1: primase makes a primer (RNA)

      • Polymerase lays down a short RNA primer on the DNA template once a sufficient length of the single stranded DNA is exposed 

      • Created a pre-existing 3’ end for DNA polymerase II to add to 

    • Step 2: DNA polymerase III

      • Adds nucleotides to the 3’ end of primer

      • Extends the primer 

      • Creates and extended a DNA daughter strand 

    • DNA continues to unwinder repeating step 1 and 2 

    • DNA polymerase I replaces RNA with DNA

      • Removes RNA from the 5’ end of a fragment and adds back DNA to the 3’ end of the adjacent fragment

    • Ligase joins fragments together

      • Makes a final covalent bond between fragments 

      • Yields one, long continuous daughter strand paired to each parental strand

      • Makes a phosphodiester bond between adjacent DNA fragments

  • Leading strand

    • 3’ end towards the fork 

    • Allows for continuous synthesis of DNA 

  • Lagging strand 

    • 3’ end away from the fork 

    • Discontinuous synthesis 

    • Results in many small DNA fragments 

  • Problems 

    • RNA in the DNA 

      • DNA polymerase I 

        • Removes RNA

        • Ads back DNA 

    • DNA fragments 

      • Ligase 

        • Adds phosphodiester bond 

  • Solved 

    • Keeps expanding bubble 

    • Keeps repeating steps

    • Stops when everything is copied 

DNA repair 

  • Molecules of inheritance (a paradox) 

    • Good: easy to copy with minimal errors 

      • Information is consistent between generations 

    • Good: able to change, making new functions 

      • Potential to evolve between generations

  • Proofreading 

    • Replication errors are repaired immediately 

      • Detected by DNA polymerase III 

    • Example of fixing an error:

      • Problem: Wrong base added

      • Solution: DNA polymerase removes a base 

      • Outcome: the correct base is added 

  • Mismatch repair 

    • Errors repaired after replication 

    • Mismatch repair complex 

      • Detected by DNA methylation 

        • Cytosine methylation 

          • Temporary modification of “C” 

          • Prevents transcription of a gene

    • Example of fixing an error:

      • Problem: wrong base detected 

      • Solution: endonuclease removes the DNA from the daughter strand 

      • Outcome: the correct base is added 

  • Excision repair 

    • DNA damage (not linked to replication)

    • Nucleotide excision repair pathway 

    • Example of fixing an error: 

      • Problem: damage is detected (damaged bases or cross-linked bases)

      • Solution: damaged bases excised 

      • Outcome: complementary bases are then added 

DNA mutation 

  • Alternate base fixation 

    • Replication before repair 

    • Error becomes a permanent change 

  • Consequences of base mutation 

    • Silent mutation: 

      • Same codon, no effect 

    • Loss-of-function: 

      • Different codon, new amino acid, new shape, disables protein 

    • Gain-of-function: 

      • Different codon, new amino acid, new shape, new function 

  • Sickle cell anemia 

    • Protein mutation = cell shape change 

      • Standard cells are flexible

      • Sickle cells are not flexible 

    • Change of function mutation 

      • Standard hemoglobin forms free tetramers 

      • Sickle cell hemoglobin polymerizes in strands 

      • Single-nucleotide change 

      • Single amino acid change 

        • Ionic to hydrophobic 

Gene families 

  • Protein diversification 

    • Different versions of similar proteins 

    • Proteins coded by different genes 

    • Example: 

      • Receptor tyrosine kinases 

  • Duplication and diversification 

    • Copies of genes in a genome 

    • Mutation leads to new functions 

Central Dogma

  • Macromolecules

    • DNA → RNA → Protein

  • Process

    • Prokaryotes 

      • DNA (

      • ) → RNA (translation) → Protein 

    • Eukaryotes 

      • DNA (transcription) + RNA processing + RNA transport 

      • → RNA (translation) + Post-translation modification 

      • → Protein 

Transcription

  • Reads DNA, copying the information to a new polynucleotide (RNA) 

    • Copies the template and produces and product in the same language (nucleotides) 

  • DNA: Genes

    • “Make” RNA by transcription

    • Sequence copied by RNA polymerase

      • RNA polymerase moves downstream 

      • Makes RNA in 5’--> 3’ direction 

      • Bubble moves with the enzyme 

  • Synthesis of RNA from DNA 

    • One DNA strand is the template 

      • Strict base pairing 

      • RNA antiparallel to template 

    • Single enzyme: RNA polymerase 

      • Separates DNA strands 

      • Makes phosphodiester bonds 

    • RNA polymerase recruitment to the promoter 

      • Makes a phosphodiester bond between nucleotides

        • Connects the sugar and the phosphate backbone of the RNA molecule

          • Doesn’t need a primer; primase can be used in DNA replication 

      • TATA box in promoter (TATAA)

        • Prokaryotes: -10 bp; direct binding 

        • Eukaryotes; -25 bp; binds TFs

      • Uses U in place of T

        • Performs all of its own functions without the aid of other enzymes 

        • Primary function: joining nucleotides together by a phosphodiester bond 

    • Eukaryotic initiation 

      • Basal transcription factors 

        • Bind the TATA box 

        • RNA polymerase binds to the basal TFs 

Steps of transcription

  • Recruitment: 

    • Recruitment of RNA polymerase to the promoter

    • Gets the enzyme to sit down on the promoter 

    • Prokaryotes 

      • RNA polymerase can recognize and bind directly to the promoter

    • Eukaryotes

      • Additional proteins are required to get the binding to occur 

      • Basal transcription factors- required for transcription, called TATA binding proteins 

        • Bind the promoter DNA and the RNA polymerase 

    • Includes TATA box 

      • Where RNA polymerase interacts with DNA 

  • Initiation:

    • Other proteins must bind to the complex for transcription to begin 

    • It can be regulated by the cell to control how much RNA is made

    • RNA polymerase binds to the promoter (with the help of transcription factors in eukaryotes) and forms a complex that begins synthesizing RNA  

  • Elongation:

    • The process of the polymerase moving downstream along the DNA and adding nucleotides to the growing RNA strand

  • Termination:

    • Required to stop translation at the correct location along the gene

    • Prokaryotes

      • Terminator sequence in the DNA

      • Causes the polymerase to stop 

    • Eukaryotes

      • No direct termination 

      • Signal on the RNA that starts the next process (RNA processing)

        • First step is to cut the RNA free from the polymerase 

        • Therefore causing transcription to stop 


Termination

  • Prokaryotes use a terminator signal 

    • Instruct termination 

      • GC-rich complementary sequences 

      • Forms “hairpin” in RNA; RNA polymerase stalls

  • Eukaryotes:

    • Just start the next process

    • Polyadenylation signal (AAUAAA)

    • The enzyme cuts RNA at the 3’ end

    • RNA polymerase stalls

    • The product is pre-mRNA 

      • Needs to be processed further 

Gene structure

  • Transcriptional unit 

    • Single-strand RNA sequence matches DNA sequence (template strand)

    • “Reverse-compliment” of template 

  • Transcription initiation site 

    • +1

    • Position of the first 5’ RNA nucleotide

    • Located on the first nucleotide of the transcribed RNA

  • Promoter

    • Recruits RNA polymerase to DNA to begin transcription

    • Upstream of +1 transcribed unit 

  • Transcribed region 

    • Transcription starts where the promoter meets this transcribed region (+1 site) 

    • Copied by RNA polymerase

    • Downstream of +1 

  • Terminator 

    • Stops RNA polymerase

    • Downstream of the transcribed region 

  • Positive control of transcription

    • Involves activator proteins that increase the rate of transcription by helping RNA polymerase bind to the promoter

  • Negative control of transcription

    • Most common in prokaryotes

    • Involves repressor proteins that block transcription by preventing RNA polymerase from accessing the promoter

  • Transcription factors

    • Act as both actuators and repressors

  • Enhancers

    • Position-dependent

    • Can be distant from the promoter


Gene regulation

  • Regulatory molecules (effectors)

    • Transcription factors (proteins that bind with DNA to either help or inhibit transcription) 

      • Negative control 

        • Repressor molecule 

        • Interferes with RNA polymerase

      • Positive control 

        • Activator molecule 

        • Helps RNA polymerase

  • Prokaryotes

    • RNA polymerase 

      • Recognizes promotor 

      • Binds to the promoter 

      • Default: make RNA 

    • Negative control 

      • Default: make RNA 

      • Repressor binds to DNA → prevents transcription

        • Regulates gene expression

  • Eukaryotes

    • RNA polymerase

      • Does not recognize the promoter

      • Will not bind to the promoter

        • Individual genes are regulated by multiple control regions on the DNA in addition to the basic promoter 

      • default: no RNA 

    • Positive control

      • Default: no RNA 

      • Basal transcription factors bind DNA → RNA pol binds basal TFs 

  • Regulatory elements- eukaryotes 

    • Promoter 

      • Upstream of +1

      • Basal TF binds 

      • Recruit RNA polymerase

      • on/off switch for transcription

        • Eukaryotic RNA polymerase will only bind to the promoter in the presence of basal TFs (activators) 

    • Proximal control elements 

      • Upstream of the promoter 

      • Regulatory TFs bind 

      • Both inhibitors and activators 

      • when/where/how much 

        • Controls how much RNA is made (regulatory TFs) 

    • Enhancer 

      • Position-independent element 

      • Activators bind 

      • Supercharger of transcription

Processing of RNA

  • Prokaryotes 

    • No additional processing required 

    • Used “as is” for translation 

  • Eukayotes 

    • Modify mRNA after transcription and before translation

    • Immature RNA is “pre-mRNA”

    • Processing occurs in nucleus 

      • 3 steps: capping, tailing, and splicing

mRNA structure 

  • Mature mRNA

    • 5’ Cap

      • Modified guanine nucleotide (added backward to the 5’ end of mRNA after transcription) 

      • Purposes:

        • Protects 5’ end from hydrolysis by RNAse as enzymes that remove nucleotides from the 5’ end 

        • Helps ribosomes recognize 5’ end 

        • Aids in export from the nucleus 

    • 5’ UTR (untranslated region)

    • Protein-coding domain

    • 3’ UTR 

    • 3’ Poly-A tail (cut free from RNA polymerase at the end of transcription) 

      • 50 - 250 adenine nucleotides

      • Polyadenylation signal in 3’ UTR

      • Purposes: 

        • Protects the 3’ end from hydrolysis 

        • Aids nuclear export (of mRNA to the cytoplasm, acts as one sign that the mRNA is ready for translation) 

        • Stabilizes mRNA on the ribosome to improve translation 


RNA splicing (removing introns from the transcript and joining the remaining exons to form a continuous coding sequence)  

  • Required because most eukaryotic genes include extra information in the transcribed region as introns that do not contribute to protein synthesis and need to be removed 

  • The protein-coded information in eukaryotic genes splits into segments (exons)

    • These are then separated by non-coding segments (introns)

  • Removes introns and joins exons 

    • Cuts out the introns and ligates the remaining exons back together into a single transcript 

  • Alternative splicing:

    • Joins exons “out of order” 

    • Isoforms

      • Different versions of a protein 


Compartmentilization

  • Transcription in the nucleus 

  • mRNA processing in the nucleus 

  • Translation in the cytoplasm


Translation 

  • Process of making polypeptides 

  • Process that uses info coded in the nucleotide sequences of RNA to assemble polypeptides 

    • Language of nucleotides to the language of amino acids 

  • How is the information in mRNA translated into a peptide? 

    • 4 nucleotides to 20 amino acids? 


The Genetic Code

  • Codons 

    • Three-letter codons each represent an amino acid

    • Includes Start and Stop signals 

    • Degenerate code

  • Reading frame 

  • Three ways to “read” a DNA sequence, but only one is correct 

  • How is the correct reading form determined? 

    • Prokaryotes: 

      • Starting at an AUG next to the Shine Dalgarno sequence (AGGAGG)

      • 8 bases before 5’ ward first AUG 

    • Eukaryotes: 

      • Scan for the first 5’ AUG 


Translation mechanism 

  • mRNA 

    • Molecular program 

  • Ribosomes 

    • Molecular machinery 

    • Always start with a Met amino acid codon, AUG, on the RNA (start codon) 

  • Amino acid monomers 

    • Raw material 

  • tRNA 

    • Molecule that performs the translator function, linking codons to amino acids 

    • Represented in the cloverleaf model 

      • Nucleotide sequences along the RNA are self-complementary, such that when nucleotides hydrogen bond to each other, they fold the tRNA in a predictable manner

        • Anti-codon at one end and an amino acid attachment site at the other 

    • 20 different versions of an enzyme called amino-acyl tRNA synthase 

      • e/a with an active site suitable for using one specific tRNA and one specific amino acid as substrates 

    • Charged tRNA: an enzyme that makes a covalent bond connecting the amino acid to the tRNA 

    • Anti-codon: at the end opposite of the amino acid attachment site, that is complementary to one of the codons for that amino acid 

    • e/a tRNA will hydrogen bond to mRNA through strict base pairing 

  • Polypeptide 

    • Product 

Ligase

  • joins 2 ends together

Helicase

  • unwinds the DNA into separate strands

Okazaki Fragments

  • short, newly synthesized DNA fragments that are formed on the lagging strand during DNA replication

Origin of Replication

  • specific sequence where DNA replication begins

Primase

  • makes short RNA sequences called primers

Topoisomerase

  • break and rejoins DNA strands

DNA polymerase I

  • responsible for filling gaps in DNA and removing RNA primers

DNA polymerase III

  • responsible for replicating both the leading and lagging strands

  • had proofreading capabilities during DNA replication

Proofreading

  • Occurs during DNA replication

  • DNA polymerase III can detect and remove incorrectly paired bases by backing up and replacing them

Mismatch repair

  • Happens shortly after replication

  • Detects mismatches that escaped proofreading by identifying the newly synthesized strand via methylation patterns and corrects the error using the parental strand as a template

Excision repair

  • identifies and removes damaged bases (not necessarily mismatches) at any time, using the undamaged strand as a guide to restore the correct sequence

Silent mutation

  • A mutation that results in a different amino acid with similar functions

Gene duplication

  • Type of mutation that leads to the evolution of gene families over time

  • Gains a function

TATA box

  • specific DNA sequence found in promoters in eukaryotes

  • binding site for transcription factors and RNA polymerase to initiate transcription

Basal transcription factors

  • Proteins required for the initiation of transcription in eukaryotes that help RNA polymerase bind to the promoter

Transcription

  • Process that reads DNA and copies its information into RNA

Introns

  • non-coding sequences removed during splicing

Exons

  • coding sequences that are joined together to make the final mRNA

Elongation steps:

  • primase makes RNA primer

  • DNA polymerase III makes Okazaki fragment 1

  • DNA polymerase III detaches

  • Fragment 2 is primed

  • DNA polymerase I replaces RNA with DNA

  • DNA ligase forms between DNA fragments

  • Lagging strand is complete

Prokaryotes have:

  • DNA polymerase III

  • fast replication

  • not packaged into chromatin

  • DNA polymerase I

  • single origin

  • Promoter sequence:

    • RNA polymerase

Eukaryotes have:

  • multiple origins

  • packaged into chromatin

  • slow replication

  • Rnase H

  • DNA polymerase a, L, E

  • Promoter sequence:

    • general/basal transcription features