315: Unit 2

DNA Polymerase

uses a strand of DNA as a template, synthesizes 5’ to 3’, and needs a primer

RNA polymerase:uses a strand of DNA as a template, synthesizes 5’ to 3’, and doesn’t need a primer

How is mRNA expression controlled at initiation?

gene specific transcription factors- activators and repressors binding to mediator

How is mRNA expression controlled at elongation?

negative regulatory factors NELF (negative elongation factor) and DSIF and elongation factors

Transcriptional factors

biochemical fractionation of nuclear extracts subsequently led to the identification of these factors; required for RNA polymerase ll to initiate transcription

General Transcription Factors

generally required for transcription of all genes by RNA polymerase ll; constitute basic transcription machinery and function to recruit RNA pol ll to the promoters of transcribed genes and then initiate transcription (dogs are best friends); bind to basal promoter; control formation of the initiation complex at basal promoter

TATA Box

a regulatory DNA sequence found in the promoters of many eukaryotic genes transcribed by RNA pol ll

Core Promoter Elements

nucleotide sequences located within the promoters of many eukaryotic protein coding genes that promote transcription via binding to one or more general transcription factors

TATA Binding protein (TBP)

a basal transcription factor that binds directly to the TATA box

TBP-Associated Factors (TAFs)

a polypeptide associated with TBP in the general transcription factor TFIID

C-terminal domain

the amino acid at the C terminus of the RNA pol ll largest subunit that consists of tandem repeats of seven amino acids with the consensus sequence (Tyr-Ser-Pro-Thr-Ser-Pro-Ser)

Pre-Initiation Complex

mediator, which interacts with gene specific regulatory factors as well as RNA pol ll and general transcription factors

Basal Promoter

where polymerases begin (ex: TATA box and CAAT box)

What organizes Eukaryotic DNA?

DNA is wrapped around nucleosome core particles that have two molecules of each histone (H2A, H2B, B3, and H4) and one H1

Mediator

a complex of protein that stimulates transcription of eukaryotic protein-coding genes and allows them to respond to gene-specific regulatory factors; binds to the preinitiation complex and enfolds it in an inactive state

Where does transcription initiate?

downstream of the basal promoter

Enhancers

gene specific transcription factors a transcriptional regulatory sequence that can be located at a start site distant from the promoter/start site

How many transcription factors are needed for recruitment of RNA pol ll?

a minimum of 5 general transcription factors

Gene specific transcription factors

required to start the transcription process; bind to enhancers and mediator; determine when and where and how much is expressed

How is a preinitiation complex formed?

TFIID binds to the TATA box, initiation element, and several other sequences; TFIIB is recruited to the BRE; RNA polymerase ll and TFIIF bind; TFIIE and TFIIH are recruited; mediator binds and enfolds the preinitiation complex in an inactive state; gene specific TF/Activator binds to the mediator and activated TFIIH which has helicase and kinase activity

What does TFIID do?

binds to the TATA box, the start site, and several other sequences

What does TFIIB do?

it is recruited to the BRE

What does TFIIH do?

it has helicase and kinase activity, but it remains inactive when recruited to the GTF; it becomes active when gene specific transcription factor binds to an enhancer and mediator

What does phosphorylation of polymerase CTD do?

it recruits elongation and chromatin remodeling factors

What are the steps of initiation?

General transcription factors bind to the basal promoter, RNA polymerase is recruited, TFIIH (helicase and kinase activities) is recruited but is inactive, Mediator binds, Gene-specific transcription factor binds to an enhancer and Mediator. This activity can be inhibited by Repressors, this activates the helicase and the kinase activities of TFIIH, DNA is unwound, RNA polymerase is phosphorylated on c-terminal domain (CTD), dissociates from Mediator and starts polymerizing

Polyadenylation signals

a nucleotide sequence at the 3’ end of protein coding genes that, once transcribed into an mRNA sequence direct cleavage and release of the mRNA to terminate transcription and addition of the 3’ polyadenine tail

Activators

bind to enhancers and mediator and activate helicases and kinase activities, releasing the polymerase; stimulate transcription

Repression Domains

the domain of transcriptional repressors that mediate inhibition of target gene expression

Repressors

block the binding of the activators to enhancers/ regulatory sequences and blocks the binding sites on the mediator

Enhancer Element

where a gene-specific TF binds

Where can enhancers be located?

They can be up to 1000bp away, upstream or downstream, and forwards or backwards in orientation.

DNA Looping

transcription factors bound at a distant enhancer are able to interact with a mediator or GTFs at the promoter because the intervening DNA can form loops, stabilized by cohesin; loops restrict enhancers to interact with promoters in the same domain; chromatin within the nucleus is organized into looped domains formed by the interaction of CTCF and cohesin

Cohesin

a protein that encircles 2 strands of DNA to form chromatin loops and maintain the connection between sister chromatids

Topologically Associated Domains (TADs)

a region of DNA within a chromosome that are maintained in association with itself, ranging from 100 to several thousand kilobases in human cells; formed by intersection of CTLF and cohesin

Electrophoretic-mobility shift assay

an assay for the binding of a protein to a specific DNA sequence

Consensus Sequence

a nucleotide sequence with bases at each position that are most commonly found in known binding sites for a transcription factor

Position weight matrix

a representation of the nucleotide sequence to which a transcription factor binds in which the frequency of bases at each position of the binding site is represented by the relative size of each base

Chromatin immunoprecipitation

a method for determining regions of DNA that bind transcription factors within a cell

DNA Affinity Chromatography

a method used to isolate DNA-binding protein based on their binding to specific DNA sequences

DNA Binding Domain

recognizes a specific dna sequence; domain of a transcription factor that binds to enhancer

Activation Domain

interacts with coactivators that facilitate transcription by modifying chromatin structure; binds to mediator and other proteins that remodel chromatin

Coactivators

a protein that interacts with a transcription factor to stimulate transcription

NELF and DSIF

negative regulatory factors pause elongation after about 50 bases transcribed

p-TEFb kinase

phosphorylates and inactivates NELF and DSIF if conditions are favorable; positive elongation factor

Elongation factors

recruited to replace NRFs (highly regulated; cMyc is a major recruiter)

Myc

one of the most common genes mutated in cancer, bypassing P-TEFb

NRFs

negative regulatory factors pause elongation

How are NELF and DSIF inactivated?

phosphorylated and inactivated by P-TEFb (positive EF-kinase)

P-TEFb

positive EF-kinase that is highly regulated

What happens in elongation?

after 50 nucleotides are transcribed, NELF and DSIF bind and stop elongation; PTEPb (a highly regulated kinase) phosphorylates and inactivates the inhibitors NELF and DSIF; elongation proceeds

PTEPb

a highly regulated kinase that phosphorylates and inactivates the inhibitors NELF and DSIF

pre-RNA

the primary transcript which is cleaved to form individual ribosomal RNAs

snoRNPs

complexes of snoRNAs and proteins; mediate rRNA modifications by guiding site-specific chemical modifications and processing of precursor rRNA during ribosome biogenesis; their substrates are pre-ribosomal RNAs

SnoRNA

class of short, non-coding RNA molecules found within the nucleus of eukaryotic cells that helps process newly made messenger RNA; act as sequence specific guides to bind and base pair directly with the pre-rRNA

Small Nucleolar RNAs (snoRNAs)

are a class of small, non-coding RNA molecules found in the nucleolus of eukaryotic cells. They act as sequence-specific guides that base-pair directly with pre-ribosomal RNA (pre-rRNA) to guide chemical modifications (like methylation and pseudouridylation) and processing necessary for making functional ribosomes.

RNase P

a ribosome that cleaves the 5’ end of pre-tRNAs

Ribozyme

RNA enzyme; act as biological catalysts; splice RNA, act as peptidyl transferase, etc

mRNPs

messenger ribonucleoprotein particles consisting of mRNA associated with proteins; responsible for mRNA processing, transport, and regulation of mRNA function

RNA processing

modification of the 5’ end with the addition of the 7-methylguanosine cap- a structure consisting of a GTP and methylated sugars added to the 5’ end of eukaryotic mRNAs (initiated by guanylyl transferase); following cleavage, the 3’ end is modified by the addition of a poly A tail, a tract of around 200 adenine nucleotides added to the 3’ end; RNA splicing

5’ cap

7-methylguanosine cap added to 5’ end by guanylyl transferase; acts as a binding site for ribosomes and stabilizes RNA

3’ poly A-tail

a tract of around 200 adenine nucleotides added to the 3’ ends of eukaryotic mRNA

Introns

excised from mature RNA

Ribosomal RNA processing

cleavage- synthesis as a 45S precursor; isomerization (uriding to pseudouridine; ribose methylation

What is snoRNA’s role in pre-rRNA processing?

snoRNAs base pair with the pre-rRNAs, aligning the enzymes at the site of processing; act as molecular guides that direct chemical modifications on other RNA molecules, primarily ribosomal RNA (rRNA)

How is transcription terminated?

AAUAA o+ U- (or GU)-rich region is transcribed and recognized by processing enzymes bound to RNA pol ll; endonuclease cleaves the strand; poly-A polymerase adds the tail

Polyadenylation

the termination signal in eukaryotes

In Vitro Splicing

a key experiment in understanding pre-mRNA splicing; genomic copy of eukaryotic gene cloned into a bacterial plasmid; plasmid linearized and transcribed in vitro (bacterial RNA processing); eukaryotic nuclear extract added

Spliceosomes

a large complex of snRNAs and proteins that catalyzes the splicing of pre-mRNAs

Small Nuclear Ribonucleoprotein Particles (snRNPs)

complex of snRNA and proteins

How is pre-mRNA spliced?

First the 5’ GU splice site is cleaved and a lariat is formed, next the 3’ AG splice site is cleaved and the exon is ligated

snRNAs

U1, U2, U4, U5, and U6; have two functions in splicing that are complementary base pairing and enzymatic catalytic activity

How is the spliceosome assembled?

First, the U1 snRNP binds to the 5’ splice site by complementary base pairing. Next, the U2 snRNP binds to the branch point (U2 binds to U1). Following this, the U5 snRNP and U4/U6 snRNP enter the spliceosome. After, the U1 snRNP and U4 snRNP dissociate from the spliceosome, and then the U2, U5, and U6 snRNPs catalyze the formation of the lariat and ligation of exons.

Self splicing RNAs

the ability of some RNAs to catalyze the removal of their own introns

RNA Editing

RNA processing events other than splicing that alter the protein coding sequences of mRNAs (deamination)

What is the role of splicing factors in spliceosome assembly?

SR and U2AF splicing factors bind to sequences in exons and recruit the U1 and U2 snRNPs to the site

Nested Gene

contained within an intron of a larger host gene; transcription yields primary transcripts of both the host and nested genes which are spliced to yield host gene and nested gene mRNA

Alternative Splicing

allows exons to be joined in different combination resulting in the formation of different mRNAs

What is the final step in mRNA transcription?

the poly-A tail and cap protect mRNA, but all degrade and turn over

Why is rRNA synthesized as a 45S precursor and then cleaved to form the 18S, 5.8S, and 28S forms?

This ensures that the three forms are always present in equal amounts

What form is tRNA synthesized in?

tRNA is synthesized as a pro form that is cleaved by both a protein enzyme and a ribozyme. It is also modified (e.g. in the anti-codon) to create the mature form.

Pre-form

Contains a signal peptide at the very beginning (N-terminus); Acts as a "shipping label" to guide the protein to a specific location (like the Endoplasmic Reticulum); it is removed inside the cell during translocation across a membrane.

Pro-form

Contains an inhibitory segment that blocks the active site; acts as a "safety switch" to keep the protein turned off until it reaches its final destination; it is often removed outside the cell or inside specific storage compartments (like lysosomes).