MBB 2

DNA sequence that encodes amino acids in a protein is

called ?

but other regions of the chromosome are non-coding

and instead have ? role which acts as

DNA sequence that encodes amino acids in a protein is

called coding sequence ...

but other regions of the chromosome are non-coding

and instead have a regulatory role:

they are binding sites for proteins that perform functions

Expression of a gene relies on

proteins that recognize

regulatory DNA sequences

RNA Polymerase

binds specifically to

promoter sequence to initiate transcription, aided by

gene regulation proteins called transcription factors

(TFs) that recognize other specific DNA sites

Where are TF binding sites

often clustered in a promoter region but might be after the promoter or inside the coding sequence

Where does translation start

ribosome binds mRNA at a specific regulatory sequence

("ribosome binding site", RBS) ... translation starts at

the nearest downstream 5' aug 3' sequence,

which is the initiation (start) codon

A biologically relevant reading frame

open reading frame (ORF)

Bacterial genes are grouped in

operons

One mRNA produces multiple proteins- has their own start and stop codons

• this arrangement is called ?

polycistronic

Adaptive benefits of polycistronic arrangement

less non-coding DNA = rapidity of DNA replication

• proteins that need to work together are expressed at same time in the same amount

What are some things RNAP can’t do that DNAP can do

• no proofreading (mRNA is short-lived, not inherited)- accuracy is less important

  • RNAP can’t start from a single DNA strand

What are some things RNAP can do that DNAP can’t do

initiate transcription starting with duplex

open duplex to expose template and polymerize single NTPS without primer

act processively without any external sliding clamp

Nucleoside triphosphates are the substrate: new phosphoester linkage forms with release of diphosphate : equation

RNA (n) + NTP →←RNA (n+1) + PP(i)

RNAP in middle of its job

DNA duplex gets renatured in rear while it gets denatured in front

What enables RNAP to recognize the promoter

RNAP core binds DNA non-specifically

• requires sigma protein for recognizing promoter

core+sigma

holoenzyme

Consensus recognition

an idealized optimal sequence

• real sequences may differ with sub-optimal expression

Sigma70

housekeeping genes most often needed

Sigma32

used in heat shock response to high temperature

• for expression of proteases and chaperone enzymes

proteases

to degrade damaged proteins

chaperone enzyme

to prevent protein misfolding

6 Steps in transcription initiation process

1. RNA polymerase binds to DNA

2. Closed complex is formed at gene promoter

3. RNA poly binds to promoter in the -35—10 regions

4. The open complex is formed at gene promoter

5. DNA is partially unwound at the initiation site of transcription

6. RNA synthesis is initiated in a primer independent reaction

Open complex

binding of sigma to promoter triggers conformational change in RNAP that denatures DNA ~10 bp

Transcription: transition from initiation to elongation

open complex

• sigma factor is relased

• PROMOTER CLEARANCE

elongation complex

• elongation continues until termination

Where does transcription occur and why

occurs in bacterial cytoplasm so ribosomes can start translation on the mRNA while transcription is ongoing

Enzymes that use a polynucleotide template rely on specific sequences in the template for initiation of their function such as:

replication origin

• for DNA replication

promoter

• for transcription

ribosomes binding site and start codon

• for translation

In bacteria initiation of RNA polymerase is guided by

regulatory sequences recognized by a sigma factor that later leaves during promoter clearance

DNA in human chromosomes is compacted by

supercoiling in nucleosomes with higher coiling levels forming heterochromatin that suppresses transcription

DNA is noncoding

with structural or regulatory purposes and introns that interrupt coding sequences (exons) are removed by splicing the RNA that is initially transcribed

Eukaryote

human cell

proteins made in cytoplasm but DNA in nucleus

• both replication and transcription happen in the nucleus, but RNA is exported to cytoplasm for translation

Why do eukaryotic cells have multiple internal compartments

they evolved from bacteria that invaded the cell

Prokaryotes

DNA and RNA and proteins are all made in the cytoplasm

Histones = DNA

a nucleosome

• fundamental packaging unit for eukaryotic DNA

Histone core

octamer

2 copies of each subunit (H2A, H2B, H3, H4) for 8 subunit total

Which histone binds outside the octamer

H1

Beads on a string

linker DNA between core particles

• arrangement leaves space for enzymes to access individual nucleosomes for replication and expression

Do prokaryotes have histones or nucleosomes

no

Are histone-DNA interactions sequence specific

histones proteins associate with the backbone of any DNA duplex this not specific for a sequence

The 3 hierarchical chromatin structure in the chromosome

1. Chromatin

• DNA in a cell is always packages with protein

• Duplex is supercoiled by nucleosomes

2. Euchromatin

• varying degree of access for transcription

• typical in interphase

3. Heterochromatin

• denser in light microscope

• no access for transcription

• complete compaction only for cell division - mitosis

What DNA is always in heterochromatin

Centromere and telomere are used for physical structure

• not for coding - sequence is repetitive

Centromeres in heterochromatin

combines with proteins in a kinetochore

• will be grabbed for pulling sister chromatids apart during mitosis

Telomeres in heterochromatin

combine with proteins to protect strand ends

Introns

non-coding DNA regions that interrupt genes

Each gene is divided into

coding segments called exons that are separated by non-coding introns

How are introns removed

splicing process

Introns facilitate

gene variation at RNA and DNA levels

Exon shuffling

can occur in chromosome exons rearranged into different order

introns that interrupt coding sequences are removed by

splicing and a 5’cap and 3’ poly-A tail are covalently attached

Eukaryotes have three RNA polymerases in the nucleus

RNAP II: dedicated to mRNA

RNAP I: for rRNA

RNAP III: for tRNA plus some rRNA other non-coding RNAs

All RNA species must be exported out the the nucleus. Why?

Since aminoacyl-tRNA synthesis and translation all happen in the cytosol

When making mRNA, the RNAP II transcription complex must

change its structure during elongation:

covalent modifications and even protein subunits are added or subtracted

The structural changes that occur when RNAP II complex changes stricture recruit enzymes for :

the mRNA maturation process

1. 5’capping

2. splicing

3. 3’ poly A tail addition

mRNA processing by specialized enzymes: Cap

5’triphosphate at the end of pre-mRNA is recognized by capping enzymes which bind to RNAP II soon after transcription inititaion

• 5’phosphate gets linked to 5’ end of a methylated G for 5’ - 5’ triphosphate link

• NO FREE 5’ PHOSPHATE

mRNA processing by specialized enzymes: Splicing

by the spliceosome complex

• composed of protein and RNA where RNA is the catalytic component, similar to the ribosome

mRNA processing by specialized enzymes: PolyA tail

at termination of transcription polyA polymerase not RNAP II adds A nucleotides

What happens is a mature mRNA transcript loses a 5’cap or 3’tail

it is excluded from translation and degraded by exonucleases

Mature mRNA is scanned to find ribosome binding sites. How?

In the cytoplasm, mature mRNA assembles into a circular complex where both 5’ cap and polyA tail are recognized by specific proteins

What is the role of ATP hydrolysis when mature mRNA is scanned to find a ribosome binding site

ATP hydrolysis is used to pull mRNA past the ribosomal subunit until the RBS (ribosome binding site) is found

Viruses

group of pathogens

• acellular

• distinct multiplication

• no cytoplasmic membrane, cytosol, organelles

  • not capable of metabolic activity

Virion

virus in a extracellular state and not capable of reproducing

• consists of a protein coat called a capsid surrounding a nucleic acid core that contains genetic material or the viral genome

plasmids

is a circular DNA duplex that is found in a hosr cell and is copied through cell divisions

viruses

are parasitic and encode coat proteins and other enzymes that enable exiting the host cell and infecting other cells

Why do plasmids persist

it carries genes that give the host an adaptive benefit

• resistance to toxins or to antibiotic drugs

What ensures that plasmid persists over generations

expression of genes conferring fitness advantage

Why can a plasmid be viewed as selfish

its sequence is adapted to ensure its replication

What are two ways bacteria can acquire new DNA

1. Transformation: importing plasmids or linear DNA directly from outside

2. Conjugation: receiving a plasmid from another bacterium via a connecting pilus

How can new DNA fragments get incorporated into a plasmid

recombination = breaking and re-joining DNA strands

virus

parasitic infectious particle

• polynucleotide with genes but protected by a protein coat (capsid) sometimes with a lipid envelope

  • no metabolism outside the host cell

Viral genome

has a replication origin like plasmids plus genes for coat proteins and enzymes to do assumbly of new virion particles ready to exit the cell

Effects on Hosts : plasmids vs viruses

Plasmids have an adaptive strategy of giving benefit to bacteria - MUTIALISM

viruses don’t benefit the host and carry genes for proteins that

• maximize replication efficiency to out compete the host using its resources - PARASITISM

• enable exiting the host and invading a new host - INFECTION

HPV: Human papillomavirus and how it exploits three hosts

virus enters into cell and into nucleus

• invades nucleus to access DNA polymerase (replication)

• & RNA polymerase (transcription)

gets into host ribosomes in cytoplasm

• coats proteins and new virus particles exit cell

oncoproteins

cause cancer in host cells

• proteins : E5, E6, E7

SARS-CoV2

example of a RNA virus

• it encodes an RNA-dependent RNA polymerase (RdRp)

SARS-CoV2 genome is single stranded RNA - explain

For replication the genome strand is used as template for RDRP to make a complementary strand which is then used as template for RdRp to make a new genome strand

spinocerebellar ataxia

traced to mutation in a gene ATXN1 residing on chromosome 6, encoding a transcription factor called ataxin-1

Do human genomes has mostly coding or non coding DNA

non-coding DNA

Pros of high percentage of non-coding DNA

• more possibilities for regulatory sequences

larger number of transcription factor binding sites

• some DNA is used for structural purposes

centromere for moving chromosomes around the cell

Cons of high percentage of non-coding DNA

• genome replication takes longer

human genome has 50k ORI sites

• proteins recognizing specific sequences have more DNA to search though

• chromosomes occupy more space in cell

PCR

Polymerase chain reaction

• uses DNA polymerase in a test tube to exponentially replicate (amplify) a targeted region of DNA duplex

  • 
    

How is target specificity achieved by PCR

with single stranded primers and cycles of annealing and melting

The combination of what 2 things enables the diagnostic detection of RNA

PCR with reverse transcriptase

DNAP cannot recognize an isolated single stranded DNA template

it can only extend a pre existing strand called a primer that is alr paired to the template

• without primer-template annealing, replication won’t happen: this specificity is exploited in PCR

PCR starts with one duplex replication by polymerase

• Synthesize two primers: one for each duplex strand

• Heat and cool to anneal primers

  • DNAP reaction with dNTPs

two key aspects of taq DNAP

processive with a built in clamp and it is heat stable so it can withstand extreme heating

Polymerase Chain reaction: exponential growth

exponential amplification

• After 1st cycle

  • Two duplexes: each has original template with new strand

• After 2nd cycle

  • Four duplexes: two with original template

• After n cycles

  • 2^n duplexes: most are new DNA strands

SARS-CoV-2 has single-stranded RNA, not DNA!

Can it be amplified by PCR?

Yes, using a special enzyme reverse transcriptase (RT)

• RT uses RNA as a template to do transcription in reverse synthesizing a strand of complementary DNA (cDNA)

Growth hormone

protein secreted by pituitary gland into blood, critical for childhood growth and tissue healing

PCR helps get starting material for

recombinant DNA

DNA cloning

the host cells in the isolated population are clonal genetically identical to the first transformed cell selected for the desired trait

the desired gene DNA inside the plasmid has been replicated exactly as part of the host cell growth so the desired gene DNA exists as a population of molecular clones

How would we control amplification of DNA through selection

Host cells whose growth we control

Vector: plasmid that can replicate in the host containing a selectable marker gene

The vector will carry our DNA of interest inside the host

Ampillicin antibiotic is added to nutrient agar before inoculation with bacteria. Why?

provides selection for the marker gene ampR (ampicillin resistance): the bacteria can't grow unless they carry the vector with ampR

Workflow for DNA cloning

1. Insert DNA of interest with a plasmid vector to make a new recombinant plasmid

2. Transform host cells with recombinant plasmid

3. Amplify (grow) lots of host cell culture: make sure we select cells carrying the plasmid

   1. With the clonal host cell culture we can isolate lots of clone DNA of recombinant plasmid OR isolate lost of protein from expression of insert

Enzymes for recombining DNA

• endonucleases (cleave strands)

• ligases (ligate or join strands)

  • recombinases (cleaves duplexes and re-ligates their strand ends in a new arrangement)

What is an expression vector and give an example

a plasmid engineered for high expression of a protein of interest

MCS : multi-cloning site

Multi-Cloning Site

sequence designed to have recognition sequences for a variety of endonucleases or recombinases

Imagine a chemical reaction you would like to do .

We can screen genes to discover an enzyme for it!

1. Get lots of DNA fragments from wherever

2. Put fragments into host cells and let cells multiple - allow mutations if you please

3. Screen/Test many cells for desired enzyme activity

   1. Select only cells with enzyme activity their DNA codes for the new enzyme

DNA sequencing

rapid using various technologies

• lots of fragments are sequenced until redundancy of sequence data is obtained this demonstrates full coverage of the chromosome

Transcriptome

all RNA in a cell

• can be copied to cDNA using reverse transcriptase

  • sequencing this cDNA shows which genes are expressed in different cells

Protein sequencing

more complex than nucleic acid sequencing because there are 20 amino acids which must be distinguished as opposed to only 4 nucleotides

Mass spectrometry

used for protein sequencing allows for analysis of the proteome (all proteins in a cell) not only which proteins are expressed in a cell but how much of each protein is present

How mass spectrometry sequencing works

1. Vaporize protein into gas phase

2. Break peptide molecule by He atom bombardment ; arrange so that you get one breakage point for each molecule which gives ionized fragments

3. Mass spectrometer deflects fragments with an electric field sorting by mass: charge ratio

DISTINGUISH AMINO ACIDS BY THEIR MASSES