BIOC 503 - Transcription/RNA metabolism

messenger RNA

aka mRNA

encode the amino acid sequences of all the polypeptides found in the cell

transfer RNA

aka tRNA

match specific amino acid to triplet codons in mRNA during protein synthesis

ribosomal RNA

rRNA

constituents taht catalyze the incorporation of the appropriate amino acids

snRNA, snoRNA

RNAs involved in RNA processing

microRNA

aka miRNA

regulate the expression of genes via binding to specific nucleotide sequences 

trancription

Synthesis of RNA using DNA as a template

• tightly regulated in order to control the concentration of each protein

single-stranded

Being ___, many RNA molecules can fold into compact structures with specific functions

• some RNA molecules can act as catalysts (ribozymes), often using metal ions as cofactors

RNA polymerase

• synthesize RNA complementary to DNA template

• sense strand of DNA has same sequence as RNA

• antisense strand is the template; complementary

In the same way DNA polymerase works, if the H bonds fit a phosphodiester bond can be made using the alpha phosphate of the ribonucleotide triphosphate

generate positive supercoils ahead, later relieved by topoisomerases

promoter

region to which RNA polymerase and other factors necessary for transcription bind

transcribed region

gene region containing protein coding information and untranslated sequences at 5’ and 3’

different

transcription and translation start at __ sites

5’ to 3’

RNA is synthesized in the ___ direction

reactive pocket

RNA polymerase binds to DNA whose strands are separated in the big ___ while nucleotide triphosphate enter via entry channel linked to that ___.

Supercoiling happens on BOTH sides, taken care of by topoisomerases.

coding strand

non-template strand that has the same sequence as the RNA transcript

template strand

DNA strand serving as a template for RNA polymerase

proofreading

BACTERIAL RNA polymerase is a large enzyme with NO ___ ability

holoenzyme

• bacterial RNA polymerase

• 5 core subunits (2 alpha to assemble complex and bind to promoter, Beta to catalyze reaction, Beta’ to bind to DNA, omega to protect from denaturation)

• additional sigma factor to guide enzyme to promoter

• LACK 3’ to 5’ exonuclease activity so higher error rate

• no proofreading activity

A-T

promoters are made of __ rich sequences favoring strand separation

=- govern efficacy of RNA polymerase binding and thus affect gene expression level

C-terminal, N-terminal

The ___ of the alpha subunits of RNA polymerase binds DNA, while the ___ of that same subunit make sure it is joint/in contact with the rest of the RNA holoenzyme

closed complex, open complex

RNA polymerase is in a ____ form when binding to promoter, but when shifting to ____ complex it triggers strand separation

NusA protein

Once transcription starts, the sigma factor attached to RNA polymerase is replaced by ___ to make it faster and more accurate

prokaryotic transcription

1- RNA polymerase core binds to DNA promoter

2- Transcription bubble forms

3-Transcription initiated by open complex form of RNA polymerase

4- promoter clearance is followed by elongation 

5- elongation continues and sigma factor replaced by NusA

5- transcription terminated, NusA dissociates, and RNA polymerase recycled

70

sigma factor ___ is responsible for housekeeping gene transcription

affinity

activators and repressors proteins change the ___ of RNA polymerase for a specific promoter, thus regulating transcription

• transcription is an energy-demanding process, thus needs to be regulated

p-independent termination

• bacterial transcription termination method

• intrinsic

• 3 Us near the 3’ end of transcript

• self-complementary regions in transcript form a HAIRPIN before 3’ end causing RNA polymerase to pause and dissociate

Long AT sequence at the 3’ end, thus creating lots of Us when transcribed which only have 2 H bonds each so it’s unstable and mechanically stops transcription

p-dependent termination

• rut site = Rho utilization element = CA-rich sequence

• rho helicase binds to rut site & migrate along mRNA to elongating RNA polymerase (uses ATP hydrolysis for the energy to do this)

• rho helicase separate the mRNA strand from the DNA template strand

RNA polymerase I

EUKARYOTIC RNA polymerase synthesizing pre-ribosomal RNA (5S, 18S, 28S rRNA precursors)

RNA polymerase II

EUKARYOTIC RNA polymerase responsible for mRNA synthesis

• fastest

• inhibited by alpha-amanitin

• CTD = carboxy-terminal domain which needs to be phosphorylated to initiates transcription

• 12 subunits

• relies on protein-protein contact (transcription factors)

RNA polymerase III

EUKARYOTIC RNA polymerase making tRNAs and some small RNA products

mitochondria

__ have their own RNA polymerase

TATA box

main EUKARYOTIC promoter recognized by RNA polymerase II

Pre-initiation complex

aka PIC

• formation of PIC is rate limiting step of EUKARYOTIC transcription

• RNA polymerase II

• 6 Transcription factors: TFII-A, TFII-B, TFII-D, TFII-E, TFII-F, TFII-H

ALL OF THAT FORMS CLLOSED COMPLEX

TATA binding proteins

initiate association of RNA polymerase to promoter

• part of multisubunit complex TFIID (transcription factor)

TFII-H

transcription factor helping RNA polymerase start transcription

• HELICASE ACTIVITY unwinds DNA

• KINASE ACTIVITY phosphorylates polymerase at CTD changing conformation and starting transcription

TBP associated factors

aka TAFs

TBP + TAF = TFIID

EUKARYOTIC TRANSCRIPTION

1- RNA polymerase II recruited to DNA by transcription factors

2- transcription bubble forms and open complex formation 

3- the CTD is phosphorylated during initiation & RNA polymerase escapes promoter

4- transcription elongation aided by elongation factors after TFIIE and TFIIH dissociates

5- Elongation factors dissociate, CTD dephosphorylated, transcription terminates, process facilitated by termination factors

Elongation factors

• ELL

• pTEFb

• TFIIS

• Elongin

nucleotide-excision repair

TFIIH also has a role in ____ (NER) so tramscribed genes are more actively repaired than silent genes

RNA polymerase inhibitors

• actinomycin D & Acridine (intercalate in DNA)

• rifampicin (binds to B subunits of bacterial RNA polymerase)

• alpha-amanitin (blocks RNA polymerase II and III of predator but leaves mushroom’s own machinery alone)