Unit 2

co-transcriptional translation

simultaneous transcription and translation in only prokaryotes

Why does co-transcriptional translation only happen in prokaryotes?

because prokaryotes do not have a nucleus, so when mRNA is made it is already in the presence of ribosomes so translation can happen when transcription is still going

template strand

used for transcription (can be either strand)

How is RNA polymerase different from DNA polymerase in how they go directionally?

different genes can be transcribed by RNA Pol in different directions along DNA

splicing

removal of introns in nucleus (RNA processing) before translation can occur

What type of cells do splicing?

Eukaryotes

transcriptional start site

(+1) where transcription begins and indicates 1st transcribed nucleotide

How is transcriptional start site indicated on a model?

a hooked arrow

upstream vs downstream

upstream is before the gene and downstream is after the gene

What does labeling the transcriptional start site +1 do for communicating about nucleotides

allows you to talk about nucleotide positions specifically

5’ UTR

an untranslated region before the start codon that does not become a protein that is necessary for ribosome to position itself

3’ UTR

untranslated region after stop codon that is necessary for cell to degrade mRNA after protein production

ssRNAP (single subunit RNA Pol) and where it is found in

one polypeptide only

found in bacteriophages, eukaryotic mitochondria, eykaryotic chloroplast

bacterial RNAP

multiple subunits

1. alpha subunit (2)

2. beta subunit (1)

3. beta prime subunit (1)

4. omega subunit (1)

5. sigma factor (1)

What RNA polymerases make up the core enzyme?

alpha subunit (2)

beta subunit (1)

beta prime subunit (1)

omega subunit (1)

What gets added to the core enzyme to make the holoenzyme?

sigma factor (1)

Why does it not matter where the sigma factor goes after the holoenzyme begins transcription?

because the core enzyme can take over whether the sigma factor stays or leaves

bacterial core enzyme

has a high overall affinity for DNA but it binds promiscuously, so it does not bind effectively to the promoter region to initiate transcription

RNA Holoenzyme

has a low overall affinity for DNA but a high affinity for -10 and -35 sites so it recognizes transcription start sites efficiently (gets it started)

Rifampin

good bacterial antibiotic by binding to the beta subunit and blocks RNA synthesis

What do RNA and DNA polymerase require as a cofactor?

Mg²+

What is the most abundant RNA

ribosomal RNA

RNAP I

synthesizes ribosomal RNA (5.8s, 28s, 18s)

RNAP II

synthesizes mRNA, miRNA, snRNAs, and lncRNAs

RNAP III

synthesizes tRNA and 5s rRNA

RNAP IV and RNAP V

synthesizes siRNAs (only in plants)

lncRNAs

long non-coding RNA that is never translated into proteins

What are two examples of lncRNAs

Xist RNA and Tsix RNA

Xist RNA

turns off all but one X chromosome

Tsix RNA

represses Xist RNA

What is unique about Xist and Tsix?

they are on the same gene but they overlap in opposite directions

siRNAs

small interfering RNAs that are only in plants and lead to degradation of mRNA

How does bacterial RNAP and eukaryotic RNAP differ?

bacterial RNAP has one enzyme doing all the work while eukaryotic RNAP has enzymes specializing in function

miRNA

degrades mRNA

promoter

region upstream of +1 site where RNA Pol binds to initiate transcription in prokaryotes and eukaryotes

Why are protein/DNA interactions specific?

because most proteins will only interact with one consensus site

Consensus sequence

sequences that are next to the gene with a predictable set of nucleotides that serves as a binding site for a protein (specifically trans-element and transcription factor)

What is another name for consensus sequence?

cis-elements

-10 consensus site

TATAAT

-35 consensus site

TTGACA

Where are the -10 and -35 consensus sites located?

In the promoter region in bacteria

What are the steps for initiation of prokaryotic transcription?

1. RNA Pol Holoenzyme binds to dsDNA promoter region

2. DNA unwinds around -10 region to form transcription bubble

3. Once a +1 site has been located, nucleotides are added to the 3’ end

4. DNA unwinds at the front of the transcription bubble and rewinds back up after it has been transcribed

Bacteria translation rate

15 amino acids per second

Bacteria transcription rate

45 nucleotides ~ 15 codons per second

Why is it beneficial for the RNA to be displaced as DNA rewinds in prokaryotic transcription?

because it allows ribosomes to translate mRNA during the process of transcription

What are the two types of termination of transcription in prokaryotes?

intrinsic (Rho independent) or Rho dependent

What is intrinsic termination?

requires a specific sequence that creates a secondary structure in the RNA

secondary structure and example

single RNA strand base pairs with itself

tRNA

What are the steps for intrinsic termination?

1. template strand of DNA contains inverted repeats followed by a string of 6 adenine nts

2. inverted repeats are transcribed into RNA followed by 6 uracil nts

3. the string of U’s that is transcribed causes the RNA Pol to pause

4. the inverted repeats in the RNA fold into hairpin loop which destabylizes the DNA-RNA hybrid and causes RNA to detach from template

Why does the string of U’s being transcribed cause the RNA Pol to pause and become unstable?

because RNA Pol requires a stable DNA/RNA hybrid and going from transcribing lots of G’s and C’s in the inverted repeats to transcribing lots of U’s makes it unstable

What does intrinsic termination look like in the DNA/RNA sequences?

There are lots of G’s and C’s in the inverted repeats with a gap between them and then in DNA there are 6 adenine nts and in RNA there are 6 uracil nts following the inverted repeats

What makes the hairpin loops so strong and permanent?

the inverted repeats are mostly G’s and C’s so there are 3 hydrogen bonds between them (mostly)

What is Rho-dependent termination?

requiring Rho hexamer and enzymatic activity to detach RNA strand from DNA strand

Rho hexamer

6 Rho proteins that separates the H-bonds between 2 strands of nucleic acid (helicase activity)

• ATP dependent

rut

Rho-utilization site that is a sequence on RNA that Rho binds to and moves towards the 3’ end

What is said to make the Rho hexamer bind to rut?

it is C-rich, but it is not very well-defined

What are the 2 hypotheses for Rho-dependent termination?

1. Rho hexamer binds to rut

2. Rho binds to unbound RNA after translation stops

Describe the theory about rut in Rho-dependent termination

Rho hexamer binds to rut before sliding toward the 3’ end of RNA to separate the RNA and DNA strands

Describe the unbound RNA theory about Rho-dependent termination

Rho hexamer binds to unbound RNA after ribosomes meet a stop codon and terminate translation on RNA strand

Why is the transcriptional start site different from the translational start site

because there is 5’ and 3’ UTR of RNA that does not get translated

transcription factor

protein that binds DNA (at a consensus site) and influences the rate of transcription

What is another name for a transcription factor

trans-element

specific transcription factor

allows unique transcriptional events and allows RNAP II to locate the correct gene that should be transcribed in the correct cell at the correct time

basal transcriptional factors (general TFs)

required for initiation of transcription by RNAP II because they bind to consensus sites in the promoter region and recruit other general TFs

What is the difference between specific TFs and general TFs

general TFs are needed by RNAP II regardless of what gene or where it is and specific TFs are specific to certain cells

mediator

interaction between specific transcription factor and basal transcription factors

Why is a mediator not a TF

because it does not bind to DNA

How can the promotor region and the core promoter be used?

to manipulate gene expression by clipping the promoter regions/consensus sites and putting them in other genes in a medical interventionist way

dimer TF

binds to 2 consensus sites at the same time which adds complexity and specificity

Parts of a transcription factor

1. DNA binding domain

2. Interacting domain (activation domain)

DNA binding domain of transcription factor structure

usually just one per TF and it is the domain that binds DNA

Interaction domain (activation domain) of transcription factor structure

interacts with other proteins (ex: mediators, RNAP II) to modulate transcription

domain

designates a specific region on a protein

-25 consensus site in eukaryotes

TATAAA

What is an example of diversity between organisms in TF structure and how they bind to DNA?

helix-turn-helix structures are found in many DNA binding domains of TFs in fruit flies

Why is chromatin remodeling needed in eukaryotes?

because TFs cannot interact with condensed DNA (30 nm fiber) because there is nowhere for them to bind

Why is chromatin remodeling only in eukaryotes?

because prokaryotes do not have histones

Chromatin remodeling

moving or removing histones to make room for transcription factors

chromatin

DNA and associated proteins (usually histones)

Why is DNA so tightly wound around the hetero-octomer that it requires chromatin remodeling?

because DNA is (-) and histone tails are (+), so they bind together tightly

closed chromatin

region of low/no transcritpional activity due to TF not having room to bind to DNA

What is another name for closed chromatin

heterochromatin

How would you describe closed chromatin gene expression?

gene expression is off

Compare the histones of closed and open chromatin

closed chromatin has hypoacetylated histones and open chromatin has hyperacetylated histones

hypoacetylated histones

histones do not have acetyl groups

open chromatin

region is available for TF binding

How would you describe gene expression in open chromatin?

gene expression is on

What is another name for open chromatin?

euchromatin

What are the two termini of a polypeptide chain?

amino terminus (NH3) and carboxyterminus (COO-)

What region of the histone is available for chemical modification?

the tails

What are the two sections of a histone?

globular section (ball) and the tail

What types of mRNA processing happens in eukaryotes?

capping, splicing, and poly-adenylation

Describe the process of capping in mRNA processing

one of the 3 phosphates at the 5’ end of the mRNA is removed and a guanine (and its phosphate) is added by a 5’-5’ linkage then several methyl groups are added to the first few nucleotides

When in the process of transcription does capping occur?

after just a few nucleotides have been transcribed

What are the functions of capping?

1. adds stability to mRNA

2. allows ribosome binding so the ribosome will recognize it

What enzyme is involved with capping?

guanylyl transferase

What does gunalylyl transferase do?

takes guanine from one place to the 5’ end of mRNA

Why is there 3 phosphates at the 5’ end of mRNA before capping occurs?

because another nucleotide has never been added to it, so there will still be 3 phosphates from the NTP

What makes mRNA that has been capped look unique?

it has a unique linkage and several methyl groups

splicing

removal of introns