BIOL 160 Exam 2 Lecture Notes

nucleic acids

• essential to all known forms of life

  • DNA, RNA

macromolecules (Nucleotide)

• composed of monomers of nucleotides, which are made of three components: a 5 carbon sugar (deoxyribose), a phosphate group, and a nitrogenous base

DNA polymerization

• nucleotides bond together by dehydration reactions

• result of polymerization is a single strand of DNA with two different ends

purines

adenine and guanine

pyrimidines

cytosine and thymine

DNA

• a long double-stranded molecules that curves into a helix (ladder with nitrogen bases making the rungs)

• 2 nucleotide strands coiled around each other form a double helix

DNA structure

• because of base pairing, one strand determines the sequence of the other strand of DNA

• two strands of DNA are complementary, which allows for precise duplication of DNA during cell division

• antiparallel - head of one strand is laid against tail of other strand, which is important in replication (two strands replicate differently)

RNA

• single-stranded and have U in place of T

• coverts genetic information into proteins: mRNA, tRNA, and rRNA

science of heredity

• study of genes and how they carry the information that makes us

• how that information is replicated and passed on to the next generation

• how they make all the proteins we need for life

genome

the genetic information in a cell (chromosome and plasmid)

chromosomes

• DNA containing structures that contain all of our genetic information in the form of genes

genes

segments of DNA that code for a functional protein or RNA

eukaryotic chromosome

• linear

• 2 copies - diploid

• 1 copy - haploid

• linear strands of DNA are wrapped with histone and other proteins to form chromosomes

• there are non-doing regions on the chromosomes

• enclosed within a nucleus

bacteria chromosome

• in cytoplasm (no nucleus)

• circular

• single copy

genotype

genetic makeup of an organism and its potential properties

phenotype

• observable traits and characteristics

  • actual expressed properties

  • determined by the genotype

genetics

while the genotype of a cell remains constant, the phenotype may change in response to environmental signals (e.g., changes in temperature or nutrient availability that affect which genes are expressed

genomics

the sequencing and molecular characterization of genomes

• first complete bacterial genomes were published in 1995

• hundreds of bacterial genomes have been fully or partially sequenced

human genome project

sequencing of the entire human genome (1984-2003) completed

genetic code

• the linear sequence of the bases provide the information that tells the cell what proteins to make

• set of rules by which our DNA is translated into proteins

central dogma

describes the flow of information from DNA to RNA to protein:

• DNA contains all the information needed to make proteins

• DNA is transcribed into mRNA

• mRNA is the messenger of information

• mRNA is translated into proteins

how we go from information in a gene to a protein

proteins

• the functional units of life (structure, carriers, and enzymes)

Why is DNA replicated?

DNA is replicated to pass on the information

replication

when one cell divides into two, DNA must be replicated faithfully for both daughter cells

• remember DNA is a double strand so looking at the dsDNA in the parental cell, each original (“old”) strand is a template that produces two new complementary strands, and each daughter inherits one new strand and one old strand (semi-conservative replication)

semi-conservative replication

each daughter inherits one new strand and one old strand

replication steps

• starts: origin of replication (one or multiple)

• steps: unwind, unzip, prime, replicate

• enzymes: DNA gyrase, DNA helicase, primase, DNA polymerase, DNA ligase

DNA gyrase

relaxes supercoiling of DNA

DNA helicase

• unwinds DNA helix

  • opens dsDNA at origin of replication & along the replication fork

primase

reads opened strands and is a type or RNA polymerase which synthesizes small RNA primers complementary to the ssDNA to start the DNA replication

DNA polymerase

• an enzyme that synthesizes (reads) new/opened strands

• need a double-stranded segment to add nucleotides (hence the RNA primer)

• polymerase can only add nucleotides to 3’ end. So, the newly synthesized strand only elongates/grows 5’ to 3’

direction of replication is

bidirectional

Overview

ligase

joins together the okazaki fragments

replication steps

1. starts at origin of replication

2. DNA helicase separates the two strands, forming a replication fork

3. primase lays down RNA primers

4. DNA polymerase uses the primer to synthesize the two strands

   • the synthesis of the new DNA on the leading strand is continuous in a 5’ to 3’ direction

   • the synthesis of new DNA on the lagging strand is done in short segments called okazaki fragments

5. the RNA primers are removed and replaced with DNA

6. DNA ligase joins all the DNA fragments

replication enzymes

replication is amazingly accurate

• error rate is 1 in 10 billion bases

• proof reading by DNA polymerase not only incorporates nucleotides but also with every NTP added check to make sure there is the correct base pairing A-T, C-G and fixes any mistakes.

differences between prokaryotic and eukaryotic DNA replication

how do circular chromosomes divide?

enzyme topoisomerase separates the two loops

Transcription

• from DNA to RNA

RNA structure

• bases: cytosine, guanine, adenine, and uracil

• sugar: ribose

• phosphate groups

types of RNA

• necessary for protein synthesis

• rRNA, tRNA, and mRNA

rRNA

ribosomal RNA part of the structure of ribosome

tRNA

transfer RNA functions as helper to bring correct amino acids to ribosomes to build new proteins

mRNA

messenger RNA carries the code from DNA to ribosomes where proteins are made

transcription

• synthesis of complementary strands of RNA from DNA template

• DNA cannot be read directly by the protein-making machinery. Thus, intermediary is needed - mRNA

• RNA polymerase uses DNA as a template and copies/transcribes the information into mRNA

• transcription only reads individual genes, not the entire genome

• RNA polymerase uses one of the DNA strands (the template/antisense strand) as a template to make a complementary RNA molecule in a 5’ to 3’ direction

steps of transcription

1. initiation - RNA polymerase binds a specific site on DNA called the promoter

   • promoter is a sequence of DNA that is recognized by the RNA polymerase - specific to that polymerase

2. elongation - RNA nucleotides are added

3. termination - RNA synthesis continues until RNA polymerase reaches a site called a terminator

translation

• expressing proteins

• the process where the ribosomes read the mRNA sequence and make a protein based on the sequence

genetic code

• mRNA stores the information about which amino acids need to get incorporated into polypeptide chain to make a protein in the form of codons

• codons are groups of 3 nucleotides that code for a particular amino acid

• is redundant; a single amino acid can be represented by more than one codon

Redundancy

there are 64 possible permutations, or combinations, of three-letter nucleotide sequences that can be made from the four nucleotides

• protects cells from genetic changes (mutations)

• 61 codes for AAs

• 1 - start codon

• 3 nonsense codons code for a stop codon which signals the end of a protein molecule

protects cells from genetic mutations.

codons

groups of 3 nucleotides that code for a particular amino acid

anticodon

• tRNA molecule helps decode a messenger RNA (mRNA) sequence into a protein

• can recognize and bind to the complementary mRNA codon.

• Each tRNA has its corresponding amino acid attached to its end.

shine-dalgarno sequence

• ribosomes bind mRNA at the shine-delgarno sequence and read the codons sequentially inserting the appropriate amino acid

• ribosome binding site

• translation starts at the AUG start codon and stops at the stop codons

Steps of translation

1. components needed to begin translation come together

2. on the assembled ribosome, a tRNA carrying the first AA is paired with he start codon on the mRNA. The place where this first tRNA site is called the P site. A tRNA carrying the second amino acid approaches.

3. the second codon of the mRNA pairs with a tRNA carrying the second amino acid at the A site. The first amino acid joins to the second by a peptide bond. This attaches the polypeptide to the tRNA in the P site.

4. the ribosome moves along the mRNA until the second tRNA is in the P site. The next codon to be translated is brought into the A site. The first tRNA now occupies the E site.

5. the second amino acid joins the third by another peptide bond, and the first tRNA is released from the E site.

6. the ribosome continues to move along the mRNA, and new amino acids are added to the polypeptide.

7. when the ribosome reaches a stop codon, the polypeptide is released.

8. finally, the last tRNA is released, and the ribosome comes apart. the released polypeptide forms a new protein.

eukaryotic transcription and translation

• transcription occurs in the nucleus - mNA has to go from nucleus to cytoplasm

• eukaryotic genes have non coding regions called introns

• RNA polymerase transcribes a primary RNA transcript that includes these introns

• introns are removed, spliced out, in a process called splicing to make final mRNA

review of replication, transcription, and translation

regulation of genetic expression

production of protein from RNA

constitutive expression

relatively constance (housekeeping genes)

• most genes > 60%

• ex. glycolysis

regulated expression

varies under different conditions

• induction and repression

transcription

operons

a number of genes that are controlled collectively by one promoter

• occurs primarily in prokaryotes

repression

activation

lactose operon (E.coli)

• inducible operon - the structural genes are not expressed unless lactose is present

• induced in the presence of lactose

• genes that are required for the transport and metabolism of lactose

• in the absences of glucose, the lac operon allows for the effective digestion of lactose (glucose is the preferred carbon source for most bacteria)

• 3 genes in the operon: lac Z, lac Y, and lac A

lac operon

lac operon and its control elements

arginine operon

• repressible operon - the structural genes are transcribed till they are turned off

• regulated so that when arginine is present in the environment the genes for arginine synthesis are not expressed

epigenetic control

changes in the regulation of gene activity and expression that are not dependent on gene sequence. epigenetic regulation is a way to control protein synthesis by directly altering the appearance of DNA without changing its sequence

methylation

can silence a gene, affect cell development, respond to stress

post transcriptional control

regulatory mechanism that stops protein synthesis after transcription has occurred

• occurs mainly in eukaryotes

• small singles stranded RNA called micro RNA

• associates with complimentary mRNA forming double stranded RNA which is targeted for destruction

• occur during development and can account for cell to cell differentiation

in bacteria, similar short RNAs enable the bacterial cell to cope with environmental stress

Mutation

• a permanent genetic change in the nucleotide sequence of the genome of an organism

• results in genetic variability that can impact viability, function and pathogenicity

• essential to natural selection in evolution and occurs at random or in nature

• can be spontaneous or induced

spontaneous mutations

• results from errors in normal biological processes of DNA replication and/or transcription

• important for evolution as they introduce genetic variation even in organisms that replicate asexually

• occurs 1 out of every 10 billion base pairs

• e.coli can divide about every 20-30 minutes = 1 million cells in 10 hours

• E.coli at least 400 mutations will have occurred

induced mutations

caused by environmental factors called mutagens

chromosomal mutations

can lead to big effects and severe phenotypic consequences

down’s syndrome - trisomy 21

• wide range of developmental delays and physical disabilities

• approximately half the people with down syndrome will have a congenital heart defect

• duplication of 500-800 genes

• phenotype is varied mainly due to variable expression of a subset of those genes

down syndrome associated complications

cri du chat syndrom - deletion in chromosome 5

• microcephaly

• weak muscle tone

• delayed development

wolf-hirschhlorn syndrome - deletion in chromosome 4

• delayed development & intellectual disability

• seizures

• facial characteristics

jacobsen syndrome - deletion in chromosome 11

• varied symptoms

• learning difficulties and cognitive impairment

• distinctive facial features

point mutations

change in a single base pair

• base substitution

  • misense

  • nonsense

• frameshifts

  • deletions

  • insertions

• mutations can happen naturally during replication or as a result of DNA damage and improper repair

why do point mutations not always lead to phenotype?

• genetic code is redundant

• every changed nucleotide does not necessarily change the amino acid sequence of the protein silent mutation

• when the change in the sequence results in a change int he amino acid sequence missense mutation

• even missense mutations do not always translate into phenotypic or functional changes

remember

silent mutation

change in a sequence does not change the amino acid sequence of the protein

missense mutation

change in the sequence which does change the aa sequence of the protein

missense

• phenotype effects may or may not occur, depending on the specific amino acid change

neutral mutation

a missense mutation that alters the amino acid sequence of the protein but does not change its function and occurs when one amino acid is replaced by another that is chemically similar or when the affected amino acid has little influence on protein function

sickle cell anemia

a single change in the globin gene

nonsense mutation

change in the sequence introduces a stop codon which will stop translation

frame-shift mutations

• mutations occur when ntds are either inserted into or deleted from the DNA sequence

• usually, pretty severe effects because they can affect all the amino acids downstream of the insertion

• can result in the introduction of a stop codon and early stop of translation

• shift the reading frame of the genetic code so that proteins may not be properly synthesized and/or function

insertion

deletion

frame shift leading to stop

mutagens

• can cause specific base changes

• nucleoside analogs - similar to the nitrogenous bases but with slightly different base pairing

  • if present during growth, it can cause mistakes to occur during replication

  • can cause small deletions and insertions so causing frame shift mutations

radiation

• x-ray and Gamma-rays

• releases electrons that bombard surrounding cells and can cause damage to DNA
  molecules

uv radiation

causes DNA damage by causing the formation of a covalent bond between 2 adjacent thymine’s in DNA

rate of mutagenesis

identifying mutations

• mutations can be detected by selecting for an altered phenotype

• positive selection

  • mutations results in a gains of function so can be directly selected for

  • antibiotic resistance

indirect selection

where the mutation results in a loss of function, screen colonies for the desired phenotype

• loss of the ability to synthesize their own histidine

replica plating

carcinogens

• many known mutagens cause cancer

• we can use baceria to determine if something is mutageneic hence likely carcinogenic