MCB3023 Exam 2

First demonstrations of horizontal gene transfer by Griffith

S cells killed mice, R cells didn’t, S heat-killed cells didn’t kill mice, but heat-killed S cells injected with live R cells did kill mice—Griffith postulated that smth released from s cells altered phenotype of R cells to convert them to pathogenic S cells (aka natural transformation, one way bacteria can acquire new genetic material)

McLeod, McCarthy how do phenotype of cells change

purified transforming factor from S cells and determined it was mostly DNA; demonstrated ability to alter phenotype of recipient cell was not affected by treatment with proteases (enzymes that degrade proteins) or RNases (enzymes that degrade RNA)—however DNases (u should know) did destroy the transforming ability

Hershey-Chase Experiment

DNA, not protein, is the genetic material by demonstrating that DNA from bacteriophages enters bacteria and directs the production of new viruses

Watson-Crick Model of DNA

DNA consists of two chains, polymers of nucleotides. Each nucleotide consists of a 5 carbon sugar, 2-deoxyribose, a phosphate group covalently linking 3’ carbon of one 2-deoxyribose to the 5’ carbon of the next sugar, one of 4 nitrogenous bases attached to the 1’ carbon of the sugar

Purines and pyramidines

purine- A/G, double ring structure; pyrimidines- C/T, single ring structure

Chains are

complementary and run antiparallel with a 5’ and 3’ end

Bacterial chromosomal DNA

single, circular molecule; plasmids are common, no histones, some have linear chromosomes

archaeal chromosomal DNA

single circular molecule; plasmids common; histones present

eukaryotic chromosomal DNA

linear molecules that are 1n and 2n during replication cycle; rarely have plasmids (but have been identified in fungi and plants)

Genes

any segment of DNA that gets transcribed into a piece of single-stranded RNA and associated DNA regions

Messenger RNA (mRNA)

RNA molecule that is translated to produce a polypeptide

ribosomal RNA (rRNA)

structural component of ribosomes

small/micro RNA (sRNA)

small non-coding RNA molecule that often affects gene expression by interacting with existing mRNA

transfer RNA (tRNA)

involved in translation, charged with amino acids; RNA molecule that delivers an amino acid to the ribosome for incorporation into a growing polypeptide during translation

Semiconservative DNA replication

one strand of original DNA molecule is conserved in each of the newly formed double-stranded molecules, preserves the sequence of the baes

oriC

the origin of replication on the chromosome, where DNA replication begins in bacteria

DnaA

A DNA- binding protein produced by the dnaA gene, proposed initiator, and DNA replication will not begin without it

ter site

a series of termination sequences that exist on the chromosome opposite of oriC, a Tus protein binds to one of the sites and stops the forward progress of one of the replication forms

Tus protein

binds to ter site and stops progress of replication forks

Topoisomerase II

forms a transient double-stranded break in the DNA molecules allowing the two circles to become disentangles

DNA polymerase I

removes the RNA primer using a 5’ exonuclease activity and fills in the resulting short gap

DNA polymerase III

adds a deoxynucleotide triphosphate to the free 3’ OH present on an existing nucleotide, removing 2 phosphates in the process, synthesize DNA at rate of 1000 bases per second; in lagging strand rep continues until DNA polymerase runs into an RNA primer

DNA ligase

links (or ligates) the final 5’ phosphate and 3’ OH ends; seals breaks from lagging strand

Leading strand

DNA polymerase can elongate DNA only from 5’ to 3’ direction, so only one strand of sing-stranded DNA at rep fork can be extended continuously

lagging strand

new primers must be added as helicase adds more of the DNA, discontinuous elongation

Okazaki fragments

short, newly synthesized DNA pieces that form discontinously on the lagging strand

DNA replication

Principal differences between Bacteria, Archaea, and Eukaryotes

Telomeres

a short sequence of bases repeated many times that end linear chromosomes of eukarya

Telomerase

consists of several proteins and a short piece of RNA that is complementary to the single-stranded DNA that forms at the end of a linear chromosome; interacts with the telomere so that TER extends beyond 3’ end of telomere

RNA polymerase

consists of 5 different polypeptides, synthesizes RNA from a DNA template

Promoter

DNA sequence where RNA polymerase binds to begin transcription

Sigma factor

combines more weakly with core enzyme, forming RNA polymerase holoenzyme and recognizes promoter sequences on the DNA, then sigma factor dissociates

Rho-independent termination

Transcription stops when the RNA forms a hairpin structure followed by a stretch of uracils, which destabilizes the RNA-DNA interaction

Rho-dependent termination

The Rho protein uses energy from ATP to help separate the RNA transcript from the DNA template, ending transcription

monocistronic genes

encode one protein

polycistronic genes

contains multiple coding regions and can encode multiple proteins, common in bacterial operons

Codons

sequence of 3 nucleotides in mRNA that specifies an amino acid or a stop signal during translation

transfer RNA

carries a specific amino acid to the ribosome and uses its anticodon to pair w a complementary mRNA codon

TATA box

a promoter associated DNA sequence found in many eukaryotic genes and some archaeal promoters

-35 region

A bacterial promoter region approximately 35 nucleotides upstream of the transcription start site. Recognized by sigma factors to help RNA polymerase bind the promoter.

Transcription in Eukaryotes and Archaea

TATA-box binding protein, RNA polymerase vs RNA polymerase II

TATA-box binding protein

Binds the TATA box or related promoter elements and helps recruit and position transcription machinery. Found in both archaea and eukaryotes.

RNA polymerase

Enzyme that produces RNA from a DNA template

RNA polymerase II

The eukaryotic enzyme primarily responsible for transcribing protein-coding genes into pre-mRNA. It also transcribes some noncoding RNAs.

archaeal RNA polymerase

More complex than bacterial RNA polymerase and shares important structural and functional similarities with eukaryotic RNA polymerases. Archaea use TBP and other transcription factors to recognize promoters

Translation

the process of protein synthesis that uses the mRNA sequence as a template to produce a specific amino acid sequence

Ribosomes

interact with the mRNA and read the sequence 3 nucleotides at a time

Codons

the groups of 3 nucleotides on mRNA

Anticodons

3 nucleotides complementary to a given codon that exists on tRNA

mRNA

the instructions that are read by the ribosome

tRNA

possesses the anticodons and is brought together with the codon

polypeptide

is formed by translation, a chain of amino acids

Shine-Dalgarno sequence

the ribosome binding site in bacteria, initiation of translation depends on recognition and interaction between this sequence, where translation begins

Only mRNA is translated!

yup thats it

Chaperonins

aid in the correct folding of polypeptides, like heat shock proteins, abundant in hyperthermophilic archaea, bind to stretches of hydrophobic amino acids that are expose only before the polypeptide folds

Signal peptides

short stretch of hydrophobic amino acids present on proteins N-terminus if they need to be embedded within or cross a membrane

Modes of gene transfer in microbes

Microbes can acquire genetic information from other sources, allowing them to develop new traits, such as antibiotic resistance

Transformation

cells take up free, naked DNA from its environment, can be incorporated through recombination or maintained as a plasmid

Transduction

DNA transferred from one bacterium to another by a bacteriophage (a virus that infects bacteria)

Conjugation

DNA is transferred directly between bacterial cells through cell-to-cell contact, pilus or other structure may help connect the cells

Recombination

the process in which DNA molecules exchange or combine genetic information

RecA

a bacterial protein that helps homologous recombination occur by promoting the pairing and exchange of similar DNA sequences

Natural competence

ability of certain microbes to natural take up DNA from their environment, a way of performing transformation

Bacterial genome: nucleus, chromosome, where is DNA, genome size, plasmids

prokaryotic, no nucleus, one circular chromosome, nucleoid region, small genome, plasmids are common

Archaeal genome: nucleus, chromosome, where is DNA, genome size, plasmids

prokaryotic, no nucleus, usually one circular chromosome, nucleoid region, small genome, plasmids in some species

Eukaryotic genomes: nucleus, chromosome, where is DNA, genome size, plasmids

eukaryotic, nucleus, multiple linear chromosomes in nucleus, larger genomes, and plasmids are uncommon in nucleus but may be present in organelles

Chromosome

The main DNA molecule containing many of the genes needed for cell function, growth, and reproduction

Plasmids

small DNA molecules that replicate independently of the chromosome, usually circular and double stranded in bacteria, frequently carry genes that provide advantages like antibiotic resistance

lysogenic phage DNA

DNA from bacteriophage that has become incorporated into a bacterial chromosome or is maintained w/in host, remains dormant rather than immediately destroying host

• Genotype

the genetic makeup of an organism, including alleles or mutations it posesses

• Spontaneous mutations

changes in DNA that occur naturally, without being deliberately caused by an external mutagen, can result from replication errors or spontaneous chemical changes in DNA, can be beneficial/harmful/neutral

• point mutation

change involving a single nucleotide or BP, can be silent, missense, or nonsense

• Frameshift

insertion or deletion of nucleotides that changes the reading frame of a protein-coding sequence, occurs when number of nucleotides inserted or deleted is not a multiple of 3

• Reversions

spontaneous mutation that corrects a metabolic abnormality back to the wild-type form

o Suppressor mutation

a second mutation compensates for effect of original mutation without necessarily restoring the original sequence

• Wild-type strain

a strain representing the typical or reference form of an organism in a particular context, standard traits used for comparison w mutants

• prototroph vs. auxotrophic mutant

phototrophs can synthesize all essential organic compounds it needs for growth whereas auxotrophic has mutation that prevents it from synthesizing a particular required compound

+ designation

functional or wild-type phenotype for the trait being described

– designation

mutant phenotype or loss of the specific function

What does leu+ versus leu- mean

leu+ can synthesize leucine, leu- requires leucine supplied in the medium

• Utility of mutants for science

investigate virulence and antibiotic resistance, determine function of specific genes, identify genes involved in metabolic pathways

• Methods for screening for mutants

selection, replica plating, patch plating

Selection

a method that allows cells with a desired trait to grow or survive while others do not

replica plating

method used to identify mutants with particular growth requirements, colonies from OG plate are transferred to new plates in same spatial arrangement and growth patterns are compared across diff media

patch plating

individual colonies are transferred to specific locations on one or more plates, test which strains grow under diff conditions to compare known or suspected mutants

Molecular cloning tools

plasmid vectors, restriction enzymes, DNA ligase, competent cells

plasmid vectors

DNA molecules used to carry foreign DNA into a host cell, usually contain origin of replication, a selectable marker, and a cloning site where foreign DNA can be inserted

restriction enzymes (restriction endonucleases)

enzymes that recognize particular DNA sequences and cut DNA at or near those sites, can create sticky or blunt ends, cut DNA fragments for cloning

DNA ligase

enzyme that seals breaks in the DNA sugar-phosphate backbone, joins DNA fragments together during cloning

competent cells

cells capable of taking up foreign DNA

Types of vectors

plasmids, phages, cosmids

Plasmids

relatively small DNA vectors that replicated in suitable host cells and are used to clone small or moderate sized DNA fragments

Phages

bacterial viruses that can deliver DNA into bacterial hosts and can serve as cloning vectors

Cosmids

type of hybrid cloning vector that can package DNA into phage particles and replicate as plasmids in a suitable host, clones larger DNA fragments

• Plasmid incompatibility

two plasmids may not be stably maintained together in same bacterial cell if they rely on the same or closely related replication or partitioning control systems; aka similar origins of replication

• Vertical gene transfer

transfer of genetic information from a parent cell to its descendants during cell division

• horizontal gene transfer

transfer of genetic info between organisms other than offspring inheritance, spreads antibiotic resistance

• Natural competence

the natural ability of a cell to take up extracellular DNA

• Genetic elements

Transposons

DNA sequences that can move from one location in a genome to another, can disrupt genes/alter gene expression