microbio exam 3 (start from lec. 16)

chromosome segragation in bacteria

prokaryotes segregate new replicated DNA during the replication process

par proteins

in Bacillus sp, Vibrio sp, and Caulibacter sp

dedicated partitioning systems that segregate sister chromosomes

quinolones and fluoroquinolones

bind topo IV and DNA gyrase, inhibiting their activity (bactericidal)

w/o activity of these protenis, cell is unable to unwind DNA ahead of the replication fork

existing replication forks stall and collapse

e.g ciprofloxacin and levofloxacin

eukaryotic chromosomes

contain multiple origins of replication

replication origins are activated in what?

clusters

new units are activated at diff. times during S-phase of cell cycle

gene

segment of DNA (at a specific site in the genome) that specifies the structure of an RNA molecule

codes for a mRNA which is then translated into a protein

parts of a gene

promoter, coding sequence, termination sequence

genes are organized into what?

operons

operon

unit of genetic material that functions in a coordinated manner by means of an operator, promoter, and one or more structural genes

what does a single promoter control?

expression of multiple genes

polycistronic RNA

multiple messages

bacterial RNA polymerase

5 proteins in core complex (holoenzymes:

2 (alpha)

beta

beta’

w

doesn’t stay bound to transcriptional machinery all the time: sigma factor

2 alpha subunits

interacts w/ regulatory proteins

beta subunit

catalytic activity

add nucleotides in 5’ to 3’ direction, always needs 3’ hydroxyl group to function

beta’ subunit

involved binding and staying bound to DNA

w subunit

stabilizes the complex

sigma factor

recognizes promoter region of gene

released soon after start of transcription

can have multiple sigma factors

each sigma factor recognizes what?

a specific consensus sequence

sequence in the promoter

sequences are generally at the -35bp and -10bp upstream of the transcription start site

mutation in consensus sequences downstream can cause what? ***double check answer

decrease in transcription

mutation in consensus sequences upstream can cause what? ***double check answer

increase in transcription

steps of transcription initiation

sigma factor binds core RNA pol., forming RNA pol. holoenzyme

RNA pol. binds promoter

pol. unwinds DNA at promoter → open complex

transcription begins and sigma factor is released at 10 bp

rifampin/rifampicin

bind beta subunit of RNA pol., inhibiting pol. activity

bacteriostatic, not often used on its own

termination

pol. slows at pause site, GC-rich sequence that forms stem loop

rho factor binds to mRNA

GC rich region followed by series of U residues

GC residues form stem loop structure + RNA pol. binds stem loop structure, causing a pause in transcription

rho factor

slides along mRNA to pol.

dissociates pol., mRNA off of RNA

differences btwn rho-dependent + rho-independent termination?

***** ask

which type of RNA is the least stable? (shortest half-life?)

mRNA

mRNA

encodes protein (transcription of DNA code)

rRNA

synthesizes protein

tRNA

shuttles amino acids

RNA

transcription of the DNA code

things common to both types of mRNA

5’ untranslated section, 3’ untranslated section

ribosome binding site **** (ask: does eukaryotic mRNA have a ribosome binding site?)

translation start site (start codon)

translation stop site (stop codon)

what do eukaryotes have that bacterial mRNA doesn’t?

eukaryotes cap their mRNA at 5’ (methyl guanosine cap) and 3’ (poly adenosine tail)

what is the function of a methyl guanosine cap and poly A tail?

allows a eukaryotic to assess whether an mRNA is intact before export from the nucleus and translation by the ribosome

stop codons

UUAA, UAG, UGA

wobble position

degenerate nature of the codons due to a different third base position and the same first two bases

how many codons code for 20 amino acids?

61 but 64 possible codons (3 of them are for stop codons)

start codon

AUG

how is the sequence of nucleotides in an mRNA molecule read?

5’ to 3’

a signal at the beginning of each mRNA sets the correct reading frame

do mammalian or bacterial cells have more types of different tRNA?

mammalian

anticodon loop will base pair (interact) w/ what?

complimentary mRNA codon

each tRNA, except those complimentary to the stop codon, is what?

“charged” w/ an amino acid at acceptor end (phenylalanine)

are there tRNAs for stop codons?

no

tRNA charging

carried out by a specific aminoacyl-tRNA synthetase

the enzyme recognizes tRNA structure in the D-Loop and Anti-codon stem of tRNA and links acceptor end of tRNA to the appropriate aa

charged tRNA

has amino acid

uncharged tRNA

doesn’t have amino acid

multiple aminoacyl tRNA synthetases are required to what?

charge the 20 diff. aa’s to the appropriate tRNAs

ribosomes

protein machine responsible for protein synthesis (known as protein polymerase)

2 subunits, 30S and 50S

what does the S in 30S and 50S stand for?

Svedberg units: measure of sedimentation rate

30S (small) and 50S (large) ribosome subunits

52 proteins, 3 rRNAs (mostly rRNA)

can bind 1 mRNA + 3 tRNAs

what do ribosomes facilitate?

proper reading of code

creation of a peptide bond btwn adjacent aa’s

dehydration synthesis

chemical reaction facilitated by a ribosome

peptide bond

bond linking the aa’s

nascent proteins

peptides or polypeptides

transcription creates what?

mRNA → multiple mRNAs made from a single gene

bacterial ribosome can readily assemble on start codons in interior of mRNA

bacterial mRNAs are often polycistronic. what does this mean?

encode several diff. proteins translated from same mRNA molecule

ribosomes bind mRNA while what? (in prokayotes)

while mRNA is still being created

• multiple proteins made rapidly from each mRNA

• advantage of not having a nucleus

peptide bond formation

translation of RNA to protein

initiation

elongation

termination

initiation

initiation factors (IF’s) bind ribosome to ribosome binding site (upstream of start site), known as Shine Delgarno Sequence

translation begins at AUG

elongation

polymerization, movement of ribosome along mRNA

EF-Ts, EF-Tu, EF-G bring GTP energy

termination - stop codons

releasing factors undock ribosome from mRNA

in-depth translation initiation

1. 30s subunit binds mRNA (spectinomycin reversibly interferes w/ mRNA interaction w/ 30S ribosome)

2. IF2 interacts w/ initiator tRNA

3. fMet-tRNA binds to start codon

4. association of initiator tRNA w/ 30S mRNA releases IF3 and allows IF1 to bind

5. 50S subunit enters complex, 50S binding to 30S complex and GTP hydrolysis releases IF1 and IF2

in-depth translation elongation

1. EF-Tu-GTP binds to tRNA and guides it to A site

2. once A site is filled, peptidyltransferase activity makes peptide bond btwn amino acid or peptide in P site and aa in A site

3. formation of peptide bond results in transfer of aa or peptide from tRNA in P site to tRNA in A site

4. EF-G-GTP complex binds to ribosome, causing 50S subunit to advance one codon. tRNA in A site moves into P site (translocation)

aminoglycoside antibiotics like kanamycin, gentamycin, streptomycin

thought to bind the initiation complex (30s-mRNA-tRNA) and block construction of complete ribosome

spectinomycin

reversibly interferes w/ mRNA interaction w/ 30S ribosome

tetracycline

binds the 30s subunit and inhibits binding of amino-acyl tRNA to the A-site of the ribosome

chloramphenicol

binds the 50s subunit and inhibits the peptidyltransferase activity

in-depth translation termination

1. uncharged tRNA leaves ribosome

2. stop codon on mRNA enters A site. since there is no corresponding tRNA, protein release factor (RF1 or 2) enters the site

3. peptidyltransferase is activated and releases completed protein from tRNA in P site

4. RF3 enters and ejects RF1 or RF2

5. ribosome recycling factor (RRF) and EF-G enter A site. GTP hydrolysis undocks 50S from 30S

6. IF3 enters 30S subunit to remove uncharged tRNA and mRNA

primary structure of a protein

unique sequence of amino acids

ribosome generates primary structure

secondary structure of a protein

found in most proteins, consists of coils and folds in the polypeptide chain

hydrogen bonding of polypeptide backbone

composed of beta pleated sheet and alpha helix

tertiary structure of a protein (3)

determined by interactions among various side chains (R groups → 3D)

covalent disulfide bond

quaternary structure of a protein

a protein consists of multiple polypeptide chains

alt. proteins

have 1,2, and 3 prime

only multisubunit have 4 prime

operons are organized into what?

regulons

regulon

system of genes, formed by one or more operons, that have a common regulatory element

common transcription factor protein to promote expression

how are genes in prokaryotes designated?

in italics w/ the first letter lowercase and NOT capitalized

e.g lacZ - refers to the gene or DNA sequence

how are proteins in prokaryotes designated?

capitalizing first letter of name and NOT using italics

e.g LacZ - refers to the protein encoded by the lacZ gene

why do cells not express every gene at maximal level under all conditions?

physical space limitations

energy and resources conservation

contradictory functions

most proteins are enzymes that carry out what?

biochemical reactions

constitutive proteins

needed at the same level all the time

microbial genomes encode many proteins that are what?

that are NOT needed all of the time

how does regulation of protein production and function help?

conserves cell’s energy and resources

1. post-translational regulation

2. regulation of gene expression

post-translation regulation

controls the activity of preexisting enzymes

• a protein is modified to change its activity

• very rapid process (seconds)

gene expression

transcription of gene into mRNA followed by translation of mRNA into protein

regulation of gene expression

controls amount of an enzyme

generally occurs at level of transcription by controlling the amnt of mRNA

sometimes can occur at translation level by controlling whether an mRNA is translated

slower process (minutes)

where does a DNA-binding domain fit in?

major grooves and along sugar-phosphate backbone

how do most DNA-binding proteins interact w/ DNA?

in a sequence-specific manner

interactions are btwn aa side chains and chemical groups on bases and sugar-phosphate backbone of DNA

what is the main site of protein binding?

major groove of DNA

what is frequently the binding site for regulatory proteins?

inverted repeats

what influences the binding of regulatory proteins to DNA?

small molecules

homodimeric proteins

proteins composed of two identical polypeptides

regulation of transcription typically requires what?

proteins that can bind to DNA

proteins that bind DNA can alter what?

transcription rates

1. binding event can block transcription (neg. regulation)

2. binding event can activate transcription (pos. regulation)

negative transcriptional regulation

native state of RNA allows RNA pol complex to be recruited, and transcription takes place

if repressor protein (downstream of promoter) binds to DNA, it inhibits recruitment of RNA pol, and transcription does not occur

positive transcriptional regulation

RNA pol can bind to promoter only if an activator protein binds to a site near the promoter

activator binding site is upstream of promoter

if activator does not bind to DNA, RNA pol cannot bind and transcription does not occur

negative regulator/repressor

bind to regulatory sequences in DNA and prevent transcription of target genes

most often repressors block what from binding promoter?

sigma factor of RNA polymerase

bind DNA at sequence called operator sequence

repressors can require what to repress or be released from binding site?

co-factors (ligand)

co-repressors

cofactors required for repressor binding

inducers

cofactors that relieve repression