Exam 3 - Molecular Genetics

location of DNA in prokaryotes

nucleoid (no membrane-bound organelles)

location of DNA in eukaryotes

nucleus (separates transcription and translation)

location of ribosomes in prokaryotes

cytoplasm

location of ribosomes in eukaryotes

endoplasmic reticulum (ER) and cytoplasm

how are genes organized in prokaryotes?

in operons - close together

how are genes organized in eukaryotes?

can be far apart - no operons

protein coding content on chromosome in prokaryotes

entire gene is coding

protein coding content on chromosome in eukaryotes

introns are removed and exons are coding

pre-translation mRNA processing in prokaryotes

minimal - some trimming and phosphates may be added to 3’ end

pre-translation mRNA processing in eukaryotes

• splicing of exons

• 5’-cap (methyl group added to guanine)

• 3’-poly(A) tail

are transcription and translation coupled in prokaryotes?

yes they are

are transcription and translation coupled in eukaryotes?

no they are not

bacterial operon

clusters of co-regulated (all on or off) genes with related functions

why is there a correlation between transcription and translation in prokaryotes?

ribosomes start translating mRNA before transcription even finishes, so the rate of one process directly influences the other

What happens to transcription when translation is quick?

This ribosome movement blocks Rho-dependent termination, allowing transcription to continue (positive correlation)

What happens to transcription when translation is stalled or slow?

Naked mRNA is exposed, so Rho binds and prematurely terminates transcription (negative correlation)

In bacteria, what molecule causes premature termination when translation slows?

Rho factor

promoter

DNA sequence (100-1000 bp long) upstream of structural genes - where RNA polymerase will bind to initiate transcription

operator

region of DNA between promoter and structural genes where a repressor (regulatory protein) will bind (stops transcription)

regulatory genes

encode transcription factor proteins that influence transcription rate of the operon

repressor

turns off transcription in response to external stimuli

activator

increases transcription in response to external stimuli

What 3 regions of the promoter does RNAP recognize and bind to?

-35 region, -10 region (TATA/Pribnow box), +1 region (start site)

Which RNAP subunit interacts with the -10 and -35 promoter regions?

sigma

What happens to the sigma subunit of RNAP once transcription begins?

It falls off the core RNAP.

Beta subunit of RNAP

Has the main catalytic domain - separates DNA strands and polymerizes RNA synthesis

Beta’ subunit of RNAP

helps stabilize the DNA-RNA hybrid during transcription

Alpha subunits of RNAP

involved in assembly and stability of RNAP transcription complex

Omega subunit of RNAP

stabilizes core subunits, essential for holoenzyme assembly

Sigma subunit of RNAP

essential for promoter recognition and binding (not part of core)

Transcription - Step 1 - Initiation

• sigma subunit binds to core RNA polymerase subunits

• sigma subunit binds to promoter

• beta subunit separates DNA strands at start site, creating a transcription bubble

  • attaches first nucleotide of RNA (A or G)

Transcription - Step 2 - Elongation

• Beta subunit adds nucleotides to growing mRNA strand

• sigma subunit is displaced by RNA strand and falls off.

• Core RNAP subunits move in 5’-3’ direction

  • beta’ stabilizes DNA/RNA hybrid

  • alphas bind to upstream region of DNA (UP element at -50) to help stabilize complex

Transcription elongation complex (TEC)

• DNA enters pincers of beta and beta’

• Free nucleotides enter through beta secondary channel

• beta active site = site of polymerization

• RNA exits through beta exit channel

a? (TEC)

downstream jaws

b? (TEC)

secondary channel

c? (TEC)

active site

d? (TEC)

active site channel

e? (TEC)

RNA exit channel

f? (TEC)

beta subunit

g? (TEC)

beta’ subunt

h? (TEC)

omega subunit

i’s? (TEC)

alpha subunits

Which strand is the template strand that RNAP creates a compliment of?

the 3’-5’ strand

Which strand is the RNA product identical to with U’s replacing T’s?

the coding strand (5’-3’)

Transcription - Step 3 - Termination

• polymerization continues until RNAP encounters terminator region of DNA

• RNAP pauses after transcribing string of Us

• Inverted G-C repeats bind to each other and form hairpin

• Hairpin causes RNAP to dissociate

what site does the antibiotic rifampin bind to?

the beta subunit of RNAP, just before the active site

how does rifampin inhibit transcription?

only allows 2-3 nucleotides to be incorporated into nascent mRNA before physically blocking further transformation

what is the effect of a sigma factor mutation?

RNAP may not bind to the promoter correctly, decreasing transcription initiation

what is the effect of a mutation in the beta subunit of RNAP?

can reduce transcription elongation efficiency or cause resistances to antibiotics like rifampin

how do promoter mutations impact transcription?

change how strongly RNAP can bind, altering transcription levels (strong vs weak).

RNAP moving from left to right uses the ______ strand as template

bottom

RNAP moving from right to left uses the ___ strand as template

top

why are transcription errors 3-4 orders of magnitude higher than DNA replication errors?

RNAP does not have proofreading abilities, unlike DNA pol.

polycistronic

many genes in one mRNA (prokaryotic)

monocistronic

one gene per mRNA/transcript (eukaryotic)

Messenger RNA (mRNA)

coding; encodes the amino acid sequence of a protein

Ribosomal RNA (rRNA)

non-coding; ribozyme; make up the ribosome, translates mRNA to proteins

Transfer RNA (tRNA)

non-coding; ribozyme; read mRNA sequence to deliver amino acids during translation

Micro RNA (miRNA)

non-coding; regulatory RNA; inhibits mRNA translation as a form of regulating gene expression

Small interfering RNA (siRNA)

non-coding; regulatory RNA; selectively degrades mRNA as a form of regulating gene expression (RNAi pathway)

ribozyme

RNA molecules with catalytic domains that act similar to enzymatic protein domains

RNA primary structure

single stranded chain of ribonucleotides

which RNA structure is this?

secondary (nearby areas of single RNA bind together) (hairpin)

which RNA structure is this?

tertiary (more distant areas of an RNA strand interact)

What type(s) of bonds do DNA and RNA share?

hydrogen bonds and phosphodiester bonds

structure of DNA?

double helix, antiparallel, supercoiling

structure of RNA?

varied complex tertiary structures

information/function of DNA

coding/non-coding, carries genetic information

information/function of RNA

coding = mRNA, also has varied non-coding such as ribozymes and regulatory RNA

________ are essential for transcription termination

hairpins

In RNA, what can guanine non-canonically pair with?

uracil

What is a pseudoknot?

an RNA tertiary structure; creates 3D structure of RNA and is especially important for function of non-coding RNAs

what are the types of prokaryotic RNA modifications?

• methylation

• trimming

• tRNA modifications

methylation of prokaryotic RNA

adds methyl (CH3) groups

• increases RNA stability

• can impact translation efficiency

• can induce structural changes

trimming of prokaryotic RNA

ribonucleases (RNAses) make cuts in mRNA

• can influence final RNA structure

• cuts non-coding RNAs out of longer RNA transcripts

which bacterial RNA is most heavily modified?

tRNA

prokaryotic tRNA modifications

• tRNA is cut from a longer mRNA transcript

• 2-40 modifications made depending on bacterium

what types of base modifications occur in tRNAs and why?

bases can be modified to dihydrouridine or pseudouracil to help tRNA fold correctly and function in translation

eukaryotic 5’ cap mRNA modification

capping enzyme adds 5’ 7-methylguanosine cap to prevent RNA degradation

eukaryotic RNA splicing

spliceosome proteins bind to 5’ splice site, cleaves intron-exon junction, and ligates exons.

• can create isoforms of the same gene leading to different functions

eukaryotic addition of 3’ poly(A) tail

polyadenylation factor syntheses the 3’ poly(A) tail on mRNA to increase stability

mRNA life span in prokaryotes

1-5 min

mRNA lifespan in eukaryotes

minutes to hours

Why does RNA degradation happen?

• the cellular concentration of mRNA helps govern the level of gene expression

• degradative pathways ensure mRNAs do not build up in the cell and make proteins that are not needed

ribonucleases (RNases) in RNA degradation

cleave sugar/phosphate backbone of RNA

endoribonucleases in RNA degradation

cleave backbone within an RNA strand (remove larger pieces, interior cleavage)

exoribonucleases in RNA degradation

cleave off one nucleotide at a time from ends of RNA strand (removes ends)

prokaryotic pathway of mRNA degradation

• endoribonuclease cleaves mRNA into piece with hairpin structure at 3’ end and a piece without the hairpin structure (open 3’ end)

• mRNA piece with open 3’ end gets degraded rapidly by exoribonucleases

• mRNA piece with hairpin (blocks exoribonuclease) either:

  • gets cut again by endoribonuclease

  • degraded by 5’-3’ exoribonuclease

  • undergoes polyadenylation then is degraded by 3’-5’ exoribonucleases

What is a direct method for studying RNA?

when RNA is visualized or sequenced in its native state

What are the direct methods for studying RNA?

• Fluorescence in-situ hybridization (FISH)

• Northern Blot

• Direct RNA sequencing (long-read; Oxford Nanopore / PacBio)

What is an indirect method for studying RNA?

RNA is converted to complimentary DNA (cDNA) and often requires amplification (reverse transcription)

What are the indirect methods for studying RNA?

• Reverse Transcription PCR (RT-PCR)

• Quantitative RT-PCR (qRT-PCR)

• RNA-seq (short-read cDNA sequencing; Illumina)

RNA Fluorescence in-situ hybridization (FISH)

• Tissue is embedded in paraffin in sectioned into very thin slices that can be adhered to a slide

  • additionally cells can be fixed and applied to slide

• Fluorescent probes are hybridized to tissue/cell sections

  • probes can adhere to DNA or RNA

• Slides are imaged under fluorescent microscope

What can be learned from visualizing the location of RNA?

• which cells express certain genes

  • body plan

  • RNA localization is key in development

Northern Blot

• RNA is rn on a gel

• Size-separated RNA is transferred to nylon membrane

• Gene-specific radioactive probes are applied to membrane

• Membrane is exposed to film. If the gene is expressed, radioactive probes hybridize to the membrane and expose the film.

What does reverse transcription do?

Creates double stranded DNA from an RNA template using a reverse transcriptase enzyme

• Binds to 3’ end of mRNA and synthesizes single strand of cDNA in 5’-3’ direction

• Degrades RNA strand

• Synthesizes complimentary cDNA strand to form double-stranded DNA

How does RT-PCR work?

• RNA is extracted and treated with DNase (to prevent DNA amplification) (semi-quantitative)

• cDNA synthesis - mix:

  • oligo d(t) primers (eukaryotic) or random primers (prokaryotic)

  • reverse transcriptase

  • RNA

• regular PCR proceeds with cDNA and gene-specific primers

• PCR product is visualized on agarose gel. Can estimate abundance of RNA by comparing band brightness to a control, highly expressed gene.

What is quantitative RT-PCR?

A tool that uses fluorescence to ‘count’ how many copies of RNA are amplified during each round of PCR

How does quantitative RT-PCR (qRT-PCR) work?

1. cDNA is mixed with PCR reagents (SYBR or gene-specific fluorescent probes (more expensive))

2. qPCR cycler takes a fluorescent image after each cycle

3. Fluorescence is graphed from the images as an amplification curve

• Ct or Cq value = cycle at which fluorescence raises above the background

  • Expression should always be normalized against a housekeeping gene

Can RNA be used as a template in PCR?

No, would need to make cDNA first. DNA polymerase cannot use RNA.