BIOL121: Lecture 6-7

What is the central dogma?

• genes (DNA) > transcript (RNA) > protein (function)

What step of gene expression is transcription?

• first

T/F: Transcription is guided by transcribed DNA regulatory sequences upstream of the gene

• false

• guided by un-transcribed DNA regulatory sequences

What is the core enzyme?

• RNA polymerase for RNA synthesis

What are the subunits of RNA polymerase?

• alpha alpha beta beta

T/F: Sigma factor is needed for initiation and elongation of RNA synthesis

• false

• only initiation not elongation

How does the sigma factor recognize promoters?

• recognizing promoters by binding to 10 and 35 regions of genes

Why is the sigma factor important?

• guides core enzyme/RNA polymerase to promoter to initiate transcription

What is the core enzyme plus sigma factor called?

• holoenzyme

Bacteria utilize multiple sigma factors to regulate transcription of () genes in response to () environmental conditions

• different x2

() can be seen as the “housekeeping” sigma factor

• sigma70

Initiation of Transcription

1. () binds to DNA

2. () recruits () and scans for ()

3. () unwinds DNA at ()

4. () is released

1. sigma factor

2. sigma factor, core enzyme, promotor region

3. core enzyme, promotor (open complex)

4. sigma factor

T/F: Core polymerase synthesizes RNA strand 5’ to 3’

• true

T/F: Added base is complementary to template strand

• true

mRNA has same sequence as () strand but uses U instead of T

• non-template

Rho () termination forms a stem loop causing the mRNA to break off the DNA and release the polymerase

independent

Rho () termination has the helicase bind to the mRNA, pulls itself to the paused RNA polymerase and breaks the polymerase

dependent

In Rho independent termination the () sequence forms the stem loop and the () beneath the pause site is unstable, causing the mRNA to break off the DNA

• GC

• UA

What are the 4 types of RNA?

• mRNA - messenger

• rRNA - ribosomal

• tRNA - transfer; carry information from DNA to protein

• sRNA - small RNAs; regulate stability or translation of specific mRNAs into protein

() is the decoding of RNA into protein

• translation

() is the reading of a DNA template to make an RNA copy

• transcription

Open reading frame is contained within mRNA and located between () codon and () codon

• translation start

• stop

T/F: Each transcript has one reading frame

• false

• three possible

Stop codon in () frame as start codon

• same

tRNAs have () structure and () which are base pair to codons in mRNA

• cloverleaf

• anticodon

() attach amino acid aa to tRNA to charge the tRNA

• aminoacyl-tRNA transferases

T/F: Ribosomes are composed of 2 subunits: 1 small and 1 large

True

Ribosomes bind to () mRNA and () tRNAs

• 1

• 3

Transcription and translation are () in prokaryotes

• coupled

Translation Initiation

1. Ribosome binding site () on mRNA allows binding to () subunit

2. () brings “bottom” of ribosome and mRNA together

3. () blocks () site

4. () escorts () to start codon

5. “top” () of ribosome docks to “bottom” () subunit

1. shine-dalgarno sequence, 30s

2. IF3

3. IF1, A

4. IF2, formylmethionine tRNA

5. 50s, 30s

Translation Elongation

1. () binds to () site

2. () formed between new aa and () in the P site

3. Ribosome shifts down mRNA () codon

1. aminoacyl-tRNA, A

2. peptide bond, growing peptide chain

3. one

Translation Termination

1. () on mRNA enters () site

2. () factor enters A site

3. () activated and releases () protein

4. () factor and () enter A site and ribosome falls apart

1. stop, A

2. protein releasing

3. peptidyltransferase, complete

4. ribosome recycling, EF-G

Microbial genomes are () as a result of heavy horizontal gene transfer, (), and a variety of () and () strategies.

• mosaic

• recombination

• mutagenic

• DNA repair

Microbial genomes have undergone extensive gene ()

• loss and gain

T/F: DNA sequences are static

• false

• not static

T/F: Mutations of single bases happens quickly

• false

• slow, accumulated through time

Large insertions of sequences are called () and transferred from other species is called ()

• genetic islands

• horizontal gene transfer

4 reasons why DNA sequences are not static

1. mutations of single bases

2. large deletions

3. large insertion of sequence

4. plasmids

Plasmids are () DNA, usually circular and transferred by () and ()

• extrachromosomal

• transformation

• conjugation

What are the size and copy number of plasmids?

• several thousand to million bases

• 0-100 per cell

Why is it important plasmids have their own origin of replication?

• important for bacterial adaptation to changing environments

  • ex antibiotic resistance

5 plasmid advantages under special conditions:

1. antibiotic resistance genes

2. genes encoding resistance to toxic metals

3. genes encoding proteins to metabolize rare food sources

4. virulence genes to allow pathogenesis

5. genes to allow symbiosis

What are the 3 transfer processes of horizontal gene transfer?

1. transformation

   1. free DNA taken up from environment and integrated into genome

2. conjugation

   1. cell-cell contact

3. transduction

   1. DNA transfer by bacteriophage

Who first demonstrated discovery of transformation?

• Griffith, Avery, and MacLeod

• R strain and heat killed S strain

Cell needs to be () in order for gene transformation to happen

• competent

In gene transformation, () in Gram positive bacteria takes up DNA, bacteria secretes (), and accumulation of it induces assembly of () located in cell membrane

• translocasome

• competent factor

• translocasome

In gene transformation, () induces (), which is the ability of cells to take up foreign DNA

• starvation/stress

• competence

() is the transfer of DNA from one bacterium to another, following cell-to-cell contact, often called () and initiated by a () protruding from donor cell

• conjugation

• bacterial sex

• special pilus

Sex pilus is type () secretion system

• 4

Gene Transfer: Conjugation

1. () proteins encoded on fertility plasmid or ()

2. Fertility plasmid or () is (), as it makes its own transfer machinery

3. In recipient cell, transferred DNA receives () and becomes ()

4. () cells become (), able to transfer DNA

5. Recipient is always ()

1. pilus, F factor

2. F factor, self-transmissible

3. a copy of F factor, F+

4. Female, male

5. F-

Some bacteria can transfer genes across ().

Agrobacterium tumefaciens which causes crown gall disease contains tumor-inducing plasmid that can be transferred via () to plant cells.

• biological domains

• conjugation

() is the process in which bacteriophages carry host DNA from one cell to another.

This occurs () as offshoot of phage life cycle.

• transduction

• accidentally

() transduction can transfer any gene from donor to recipient and () can transfer only a few closely linked genes between cells

• generalized

• specialized

Gene Transfer: Transduction

1. Viruses inject () into cell

2. At end of life cycle, viral DNA packaged into () before ()

3. Sometimes () by mistake

4. Virus carries ()

5. Transfers DNA to new host and brings DNA from previous host to new host

1. viral DNA

2. viral capsid, cell lysis

3. package bacterial DNA

4. host DNA - transducing particles

In specialized transduction, () is when some viruses can integrate their genome into bacterial chromosome at att sites.

When entering (), bacterial genes () to viral () sites are sometimes mistakenly picked.

• lysogeny

• lytic cycle

• adjacent

• attachment

Most foreign DNA will be degraded by () in recipient cell.

Plasmids will coexist in cell as ().

DNA can incorporate into chromosomal DNA by ().

• restriction endonucleases

• extrachromosomal DNA

• recombination

In order for foreign DNA to incorporate itself into chromosomal DNA by recombination there are 3 requirements:

• homologous DNA sequences present

• replaces variable-sized section of endogenous DNA

  • could be used to repair damaged DNA

• specific recombination proteins

As a defense against transferred DNA, bacteria adds () groups to its own DNA using () enzymes to protect () sites.

Unless it comes from similar species and has () groups protecting DNA, foreign DNA without DNA () is destroyed.

• methyl

• matching methylation enzymes

• restriction

• methyl

• methylation

As a defense against transferred DNA, CRISPR is a bacterial () viruses and viral DNA.

When infected, bacteria cut up viral DNA and insert some pieces into () to () for future infections.

• immune system against

• their own genome

• remember

As a defense against transferred DNA, (), which are the little virus bits added to bacterial genome, are transcribed then () monitors the cell for matching invading DNA.

Bacteria () matching DNAs to prevent ().

• spacers

• Cas9

• degrades

• viral infection

Mutants generate new () in population and () are heritable changes in nucleic acid bases in genome of organism.

• variants

• mutations

T/F: Mutations are common and changes the genotype of organism

• false

• rare

T/F: Mutations become fixed in populations when they are selected for by natural selection

• true

What are the 2 types of mutations and what are they caused by?

• spontaneous mutations

  • errors in DNA replication

• induced mutations

  • treating organisms with added mutagens

3 agents causing mutations:

• chemical

  • carcinogens

• physical

  • radiation/heat

• biological

  • insertion of transposons

6 types of mutations:

• point mutation

  • change in single base

• transition

  • purine to purine (A or G)

  • pyrimidine to pyrimidine (C or T)

• transversion

  • purine to pyrimidine

  • pyrimidine to purine

• insertion/deletion

• inversion

  • DNA flipped

• reversion

  • DNA mutates back to original sequence

() mutation doesn’t change the amino acid because of () of genetic code.

() mutation produces a stop codon.

() mutation leads to amino acid change.

() mutation disrupts the reading frame meaning they aren’t multiples of 3.

• silent, degeneracy

• nonsense

• missense

• frameshift

Physical mutagens such as () and () cause formation of toxic () radicals.

Radicals cause two adjacent () bases to () which prevents DNA () and gene ()

• UV, ionizing radiation, oxygen

• pyrimidine, dimerize, replication, transcription

Chemical mutagens: () are similar structures to natural bases and are incorporated during DNA replication which leads to () because of ().

() changes a bases structure and pairing characteristics.

() distort DNA to induce single nucleotide () or ().

• base analogs, point mutation, incorrect base pairing

• DNA modifying agents

• intercalating agents, insertion, deletion

T/F: Chromosomes sequences are fixed

• false

Transposons are () discovered by Barbara McClintock in ().

They are segments of DNA that can () from one place in DNA to another.

They are widely used as () in molecular genetics.

• jumping genes, corn

• hop

• mutagen

T/F: Transposons are limited to bacteria

• false

• all living things

Transposition: () cuts DNA at () repeat sequences at both ends of transposable element and target sequence of target DNA; and then () the transposable element with target DNA

• transposase

• inverted

• ligates

In () transposition, transposable element jumps from one site to another.

In () transposition, transposable element is copied and one copy remains in original site.

• nonreplicative

• replicative

() examines ability of chemical substance to cause () or strength of () tested.

It is commonly used to determine if chemical has potential to cause cancer

• Ames test, mutation, mutagen

Ames test uses () that can not synthesize amino acid ().

Plate bacteria on () with the chemical you are testing for mutagenicity.

• bacterial mutant, histidine

• defined medium without histidine

On the Ames test plate, more colonies means () mutagens and no growth means () mutagens.

• stronger

• no

Mutations in DNA repair pathways can lead to () which is the rapid accumulation of mutations.

• hypermutation phenotypes

() is the correction of mismatch by DNA polymerase III during DNA replication.

3’ to 5’ () activity that removes () during ()

• proofreading

• exonuclease, mismatch, polymerization

() is the photolyase enzyme that binds to a pyrimidine dimer caused by UV radiation and cleaves cyclobutane ring

• photoreactivation

() repair is the most common damage/mismatch repair and it cuts out () DNA and recopy remaining () strand.

• excision

• duplex

• complementary

Cut, copy, paste mechanism of excision repair

1. () removes damaged () or ()

2. () copies template to replace () strand

3. () seals nick to give ()

1. enzyme, base, string of nucleotides

2. DNA polymerase, excised

3. DNA ligase, intact, repaired DNA

Nucleotide excision repair

• recognizes damages that cause () in DNA structure

• an () removes patch of () containing damaged bases including ()

• distortion

• endonuclease, single-stranded DNA, dimers

Base excision repair

• recognizes and repairs bases that () DNA structure

• recognition at () bases

• don’t distort

• damaged

Methyl mismatch repair

• recognizes () missed by ()

• use () as an () for newer strand containing error

• mismatches, proofreading

• DNA methylation, indicator

() induces SOS regulatory system

() represses expression of SOS genes

() activated by DNA damage to inactivate ()

DNA () are error prone DNA () that repair DNA rapidly, but generate numerous mutations

• extensive DNA damage

• LexA

• RecA, LexA

• DNA mutases

() are SUPER bad for bacterial DNA because it causes chromosomal instability, problems with replication, etc

• double-strand breaks

Double strand break repair with () ends joining, it doesn’t require (), and () bind broken ends and include () and other proteins recruited to fix break

• non-homologous

• homology

• Ku-family proteins

• ligases