Micro test 3

DNA converted to mRNA via

•transcription

3 nucleotides encode specific amino acid are called

•codon code)

mRNA converted to amino acids to protein via

•translation

Genome:

complete set of genetic information

Bacteria and Archaea generally have ----- chromosome

one, -haploid - 1N)

Eukaryotes generally have a ---- of each chromosome

pair -(ex: diploid - 2N)

Where are plasmids found

-Generally found in Bacteria and Archaea

nucleoid-associated proteins (NAPs)

•aid in folding and structure through electrostatic interactions (no covalent bonding).

Structure of nucleoid organized into

into 1 Mbp macrodomains that are further divided into chromosome interacting domains

Core genome

-= set shared among all/most strains

Dispensable genome

-= set shared among a few strains

Unique genome

-strain specific set of genes

Pan-genome

The set of all genes present in all strains of a group of organisms (species) (~50% match to organism genome)

•Genotype

•Specific set of genes for a particular trait

•Phenotype

•Collection of observable characteristics - influenced both by genotype and the environment

•Cells must accomplish two tasks to multiply

1.DNA replication - DNA duplicated to pass on to progeny

2.Gene expression - Synthesis of products encoded by genes

Transcription

-DNA to RNA

Translation

RNA to protein

•DNA replication Begins at

origin of replication

origin of replication

•Specific sequence in genome at which replication begins

Bacterial chromosomes and plasmids typically have how many origin sites.

•1 - oriC

•Eukaryotes and Archaea can have how many origin sites.

many

Any DNA without origin

•will not be replicated (some plasmids)

•DNA replication is usually

bidirectional

How does Replication begins at the origin of replication

•Proteins recognize and bind to the site

•Melt double-stranded DNA

How many replication forks are created (unwound DNA)

2

terminus

replication forks Ultimately meet at this terminating site

•Replication is

semiconservative

•DNA contains one original (parental), one newly synthesized strand

The start of DNA replication is controlled by ----- and

DNA methylation and by the binding of a specific initiator protein to the origin

The oriC origin is a

245-bp sequence that includes a series of repeats

Initiations are triggered by

DnaA, a protein that increases in concentration cell during growth and promotes DNA unwinding at oriC.

DNA A binds to oriC and recruits

Helicases (DnaB). Helicases disrupt H-bonds to exposes ssDNA.

Replisome

12 proteins involved in replication.

•Two replisomes move in either direction away from the origin.

Helicase

ring encircles D N A, disrupts H-bonds and provides force to move the replisome.

Protein DnaA (replication initiation factor)

recognizes oriC sequence causing bending and separation of strands and then recruits DNA helicase and gyrase, which bind to origin

Helicase

separates DNA - ring encircles DNA, disrupts H-bonds between strands, starting at fork

Gyrase

•unwinds DNA - relieves twist generated by the rapid unwinding of double helix by transiently breaking, then resealing DNA strands.

SSB Protein

coats ssDNA and prevents the re-annealing of single strands

DNA primase

•initiates DNA synthesis

•Synthesizes small strand of RNA that serves as a primer (fragment of RNA) that DNA polymerase III attaches to in order to begin DNA replication

•Leading strand

-Synthesized CONSTANTLY as DNA polymerase III moves towards the replication fork

DNA polymerase only synthesizes

-5' to 3':

•Lagging strand

-Synthesized INTERMITTENTLY as DNA is unwound (polymerase only synthesizes 5' to 3')

Okazaki fragments

Small fragments of DNA produced on the lagging strand during DNA replication, joined later by DNA ligase to form a complete strand.

lagging starnd produces

-Produces Okazaki fragments as DNA polymerase moves away from replication fork

DNA Poly I and DNA ligase

removes RNA primer and creates covalent bond between nucleotides of adjacent fragments

A different type of DNA polymerase (DNA polymerase I

will move along replication fork removing RNA primers and replace them with DNA

DNA ligase

will join fragments together

what kind of bond does DNA ligase form

forms a phosphodiester bond between 3'-OH of growing strand and 5'-phosphate of an Okazaki fragment.

•There are as many as --- terminator sequences (ter) on the E. coli chromosome.

ten

Tus

protein (terminus utilization substance) which binds to these termination sequences (ter) and ensures that the polymerase complexes do not escape and continue replicating DNA.

chromosomes which are linked

catenated

Decatenated Chromosomes form

When toiposomerase produces a double strand break and rejoins DNA strands to form 2 daughter chromosomes

•Transcription

-Synthesizing RNA from DNA

•Translation

-Synthesizing protein from RNA

messenger RNA (mRNA)

carries the message from DNA to the ribosome

ribosome made from

rRNA

Monocistronic

-one gene for one protein

•Most mRNA transcripts in eukaryotes

Polycistronic

several genes for multiple proteins

•Most mRNA transcripts in prokaryotes

•Subunits that build a final complex protein

RNA polymerase

synthesizes single-stranded RNA

•Uses DNA as a template

•Synthesizes in 5′ to 3′ direction

•Can initiate without primer

•Binds to promoter

•Stops at terminator

promoter

•Sequence of DNA upstream of genes

terminator

•Sequence of DNA that ends transcription

RNA Polymerase is a Core enzyme composed of 5 polypeptides

•(two α subunits, β, β′, and ω); catalyzes R N A synthesis.

The sigma factor (σ)

has no catalytic activity but helps the core enzyme recognize the start of genes. Recognizes promoter

•core enzyme + sigma factor.

R N A polymerase holoenzyme

•Only the holoenzyme can begin transcription.

RNA polymerase uses

the antisense/minus (-)/template DNA strand as the template strand for transcription

RNA transcript is identical to the complement

•sense/plus (+)/coding DNA strand

•Except uracil is used in place of thymine

Transcription steps - Initiation

•RNA polymerase scans and binds to a promoter

The promoter region contains a

specific nucleotide sequence that signals where to begin transcription and which direction to synthesize

Promoter is located

•at the start of the gene.

The promoter functions

•Recognition/binding site for RNA polymerase.

•Orients polymerase

Leader sequence

•is transcribed into mRNA but is not translated into amino acids.

Shine-Dalgarno sequence

important for translation initiation. -> 30S ribosomal subunit binds to it.

•Coding region

•Begins with D N A sequence 3'-T A C-5'.

•Produces codon A U G.

•Ends with a stop codon, immediately followed by the trailer sequence which prepares R N A polymerase for terminator sequence.

Consensus sequences of sigma70 promoter

TTGACAT ------------------- TATAAT

-35 region 17+-1 bp -10 region

Promoter orientation

dictates direction of transcription and which strand is used as template

promoter location

•Found -10 to -35 bp upstream of where transcription starts

Once RNA polymerase has moved past,

sigma factor is released, another RNA polymerase can bind

The sigma factor enables

specific binding of RNA polymerase to the gene promoter

RNA polymerase then denatures (disrupts H-bonds) a short stretch of DNA exposing nucleotides of DNA to form

transciption bubbles

•Bacterial promoter features:

•35 basepairs upstream (T T G A C A)

•Sigma factor recognizes this sequence directing holoenzyme to "settle" here.

•10 basepairs upstream (T A T A A T)

•Where D N A strands start to separate.

Different sigma factors recognize

different promoter sequences in the genome

How many sigma factors does E. Coli have

7

RpoD (σ70 )

- the "housekeeping" sigma factor transcribes most genes in growing cells

RpoH (σ32)

-the "heat shock" sigma factor, turned on when bacteria are exposed to heat. Expresses heat shock proteins (ex: DNA-repair enzymes), which enable the cell to survive higher temperatures.

Where does RNA polymerase denature DNA to form transcription bubble

-10 location

Sigma factors are unique to

prokaryotes

-transcription factor B which is homolgous to sigma factor is found in.

Archea

-transcription factor II B which is homolgous to sigma factor is found in.

Eukaryotes

Nucleotides are added only to the 3' end using

•(-)/antisense strand as a template

‣Uracil (replaces thymine) with Adenine

Cytosine with Guanine

Elongation fueled by

by hydrolyzing high-energy phosphate bond from incoming nucleotide

RNA polymerase continues until it reaches a

terminator

§All bacterial genes use one of two known transcription termination signals:

1.Rho factor-dependent

2.Rho-independent (intrinsic termination)

1. Rho factor-dependent

•Relies on a protein called Rho and a strong pause site at the 3′ end of the gene

2.) Rho-independent (intrinsic termination)

-GC-rich terminator sequence results in a hairpin loop in the RNA transcript

-Causes newly synthesized mRNA to be released and polymerase to disassociate

Rho factor binds to

•to rut site on mRNA and scans until it reaches RNA polymerase, which is stalled at a pause site (specific sequence on DNA template strand)

•Rho's --- activity causes everything to disassociate

helicase

•Hairpin loop along with ------ causes newly synthesized mRNA to be released and RNA polymerase to disassociate

Nus A protein

Translation overview

•Converting the information from mRNA (strand of nucleic acids) to synthesize protein (chain of amino acids)

Messenger RNA (mRNA)

1.carries the information

Ribosomes

1.ribosomal RNA (rRNA) serves as a scaffold for ribosome

decodes m RNA