Chapter 8: RNA Synthesis and Processing

What is the first step in gene expression and the initial level at which gene expression is regulated?

transcription

What does RNA polymerase do?

catalyzes polymerization of ribonucleoside 5' triphosphates (NTPs) as directed by DNA template

How many subunits does bacterial RNA polymerase?

5

What does the alpha subunit do in bacterial RNA polymerase?

identifies the correct sites for transcription initiation

What is a promoter?

gene sequence to which RNA polymerase binds to initiate transcription

6 nucleotides long

located at 10 and 35 base pairs upstream of transcription start site

What is the functional importance of the -10 and -35 regions of promoters?

genes with promoters other than these are transcribed less efficiently (alpha subunit binds to both -10 and -35 regions)

What is done to DNA to allow transcription to begin?

polymerase unwinds 12-14 bases to form an open promoter complex

When is the alpha subunit release from the polymerase?

after the addition of about 10 nucleotides

What happens during elongation?

polymerase maintains unwound region of about 15 base pairs

Where is the polymerase active site contained?

channel between the beta and beta' subunits

RNA synthesis continues until the polymerase encounters a ___

stop signal

What is the most common stop signal?

symmetrical inverted repeat of GC-rich sequence followed by 7 A residues

What terminates transcription of the GC-rich inverted repeat?

transcription results in segment of RNA that forms stable stem-loop structure which terminates transcription

What three enzymes are involved in lactose metabolism? What does each do?

enzymes only present when lactose is present

1. beta galactosidase: cleaves lactose into glucose and galactose

2. lactose permease: transports lactose into cell

3. transacetylase: inactivates toxic thiogalactosides that are transported into cell with lactose

What is an operon?

a set of genes transcribed under the control of an operator gene

What are the two operon loci that control transcription?

1. o (operator): adjacent to transcription initiation site

2. i (not in operon): encodes protein that bind to operator

The normal i gene product is a ___, which ___ transcription when bound to o.

repressor

blocks

What is the central principle of gene regulation?

control of transcription is mediated by interaction of regulatory proteins with specific DNA sequences

How do cis-acting control elements and other proteins differ in geen expression?

cis-acting elements affect expression of genes on the same DNA molecule

other proteins affect expression of genes on other chromosomes

What's the difference between negative and positive control?

negative control: regulatory protein (repressor) blocks transcription

positive control: regulatory proteins activate transcription

Explain the positive control of the lac operon by glucose in E coli

1. presence of glucose repressions expression on lac operon

2. glucose decreases then cAMP levels increase (positive control system)

3. cAMP binds to regulatory protein catabolite activator protein (CAP)

4. CAP binds to target DNA sequence upstream which facilitates RNA polymerase binding to promoter

Eukaryotic cells have ___ RNA polymerases that transcribe different classes of genes.

3

Transcription takes place on ___ because why?

chromatin

because regulation of chromatin structure is important in regulating gene expression

What does RNA polymerase II synthesize?

mRNA

What are general transcription factors?

proteins involved in transcription from polyermase II promoters

What promoter sequence does the TATA box resemble?

the -10 sequence of bacterial promoters

How many general transcription factors are required for initiation of transcription?

5

What transcription factors bind with RNA polymerase II to form the preinitiation complex?

TFIIB, TFIIF, TFIIE, TFIIH

What is transcription factor TFIID?

multiple subunits including TATA binding protein (TBP) and other subunits bind to the lnr, DCE, MTE, and DPE sequences

What is the transcription mediator?

protein complex of more than 20 subunits

interacts with general transcription factors and RNA polymerase

What binds to nonphosphorylated carboxy terminal domain (CTD) of polymerase II and is released following phosphorylation of CTD when transcription initiates?

mediator complex

How does phosphorylated CTD facilitate mRNA synthesis and processing?

binds elongation and processing factors

RNA polymerase I transcribes ___ genes

rRNA

Transcription of rRNA genes yields a large 45S pre-rRNA that is processed to yield what?

28S, 18S, 5.8S rRNAs

Promoters of rRNA genes are recognized by two ___ which recruit ___ to form initiation complex.

transcription factors

RNA polymerase I

Genes for what are transcribed by polymerase III?

tRNA, 5S rRNA, some small RNA (expressed from three types of promoters)

What do gene transfer assays identify?

cis-acting regulatory sequences in expression of eukaryotic genes

What are enhancers?

regulatory sequences located farther away from start site

Activity of enhancers ___ (does or does not) depend on their distance from or orientation with the transcription site.

does not

How do enhancers function?

by binding transcription factors that regulate RNA polymerase (like promoters)

What is DNA looping?

process allows transcription factor bound to distant enhancer to interact with proteins associated with RNA polymerase/mediator complex at promoter

What do insulators do for the activity of an enhancer?

maintain specificity

chromosomes into independent domains, prevent enhancers from acting on promoters located in adjacent domain

What are topologically associating domains (TADs)?

discrete chromosomal domains

enhancers and promoters within a TAD interact frequently but rarely with elements in other domains

What is the main protein that binds insulators in vertebrates?

CTCF

What do transcriptional activators do? What do their two independent domains?

bind to regulatory DNA sequences and stimulate transcription

1. one region binds DNA

2. one region stimulates transcription by interacting with other proteins (ex. mediator)

What does the zinc finger DNA binding domain do?

binds zinc ions and folds into loops ("fingers") that bind DNA

What does the helix-turn-helix DNA binding domain do?

one helix contacts the DNA and the other helices lie across the complex to stabilize the interaction

What are homeodomain proteins?

proteins that are important in regulation of gene expression during embryonic development

Leucine zipper and helix-loop-helix proteins contain DNA-binding domains formed by ___ of two ___

dimerization

polypeptide chains

different members of each family can dimerize with each other (combinations can form an expanded array of factors)

Activation domains stimulate transcription by what two mechanisms?

1. interaction with mediator proteins and general transcription factors

2. interaction with coactivators to modify chromatin structure

Transcription can also be regulated at ___

elongation

What do negative regulatory factors do (NELF-negative elongation factors and DSIF)?

polymerase pauses within 50 nucleotides following initiation

What is the basic unit of chromatin?

nucleosome: 147 base pairs of DNA wrapped around two molecules of each histone H2A, H2B, H3, H4

Chromatin can be altered by ___ and ___

histone modifications

nucleosome rearrangements

What is histone acetylation?

amino-terminal tail domains of core histones are rich in lysine and can be modified by acetylation

Transcriptional activators and repressors are associated with ___ and ___ respectively

histone acetyltransferases (HAT)

histone deacetylases (HDAC)

How does acetylation increase the availability of the DNA template for transcription?

neutralizes positive charge on lysine, this relaxes chromatin structure

How is histone modified?

by methylation of lysine and arginine residues, phosphorylation of serine residues, and addition of small peptides (ubiquitin and SUMO) to lysine residues

modifications occur at specific amino acid residues in histone tails

How do histone modifications affect gene expression?

altering chromatin properties and providing binding sites for proteins (activate or repress transcription)

What are binding sites for proteins that induce chromatin condensation and formation of heterochromatin?

methylated H3 lysine-9 and -27 residues

Promoters and enhancers have distinct chromatin features, are free of ___, and are thus accessible for binding transcription factors. These regions can be digested with ___

nucleosomes

DNase (they are DNase hypersensitive sites

What are chromatin remodeling factors? What can they do?

protein complexes that alter contacts between DNA and histones

reposition nucleosomes, change conformation of nucleosomes, eject nucleosomes from DNA

Following initiation of transcription, elongation is facilitated by ___ associated with the phosphorylated ___ of RNA polymerase II

elongation factors

C-terminal domain

Elongation factors include what?

histone modifying enzymes and chromatin remodeling factors

What is epigenetic inheritance?

transmission of information that is not in the DNA sequence

How does histone modification lead to stable patterns of modified chromatin?

modifications can regulate one another

What is DNA methylation?

methyl groups are added at the 5-carbon position of C that precede G (CpG dinucleotides)

controls transcription in eukaryotes, correlated with transcriptional repression

mechanism for epigenetic inheritance

What is genomic imprinting?

expression of some genes depends on whether they come from the mother or father

What do miRNAs do?

act by RNA interference pathway to inhibit translation or induce degradation of homologous mRNA

What do noncoding RNA molecules do?

form complexes with proteins that modify chromatin and recruit these complexes to their sites of transcription (regulates expression of neighboring genes)

How do IncRNAs work?

repress target genes by forming complexes with Polycomb Repressive Complex2 (PRC2)

associate with different chromatin-modifying enzymes and function as repressors or activators

act in trans by recruiting chromatin-modifying complexes to distant target genes

___ are used immediately for protein synthesis while still being transcribed

bacterial miRNA

How are ribosomal RNAs derived in eukaryotes and prokaryotes?

Eukaryotes: four rRNAs 18S, 5.8S, 28S, 5S (5S rRNA transcribed from separate gene)

Prokaryotes: cleaved to form three rRNA (16S, 23S, 5S)

What is a ribozyme?

an enzyme in which RNA rather than protein is responsible for catalytic activity

tRNAs start as long pre-tRNAs in prokaryotes and eukaryotes. Processing of the 5' end of the pre-tRNA is cleaved by the enzyme ___

RNase P

In eukaryotes, pre-mRNAs are extensively modified before ___

export from the nucleus

The 5' end of the transcript is modified by addition of a ___. What does it do?

7-methylguanosine cap

the cap stabilizes RNA and aligns it on the ribosome during translation

At the 3' end, a poly-A tail is added by ___. Signals for ___ include what?

polyadenylation

signals include conserved hexanucleotide and a G-U rich downstream sequence element

Recognition of the polyadenylation signal leads to ___, ___, and ___ of the mRNA

termination of transcription

cleavage

polyadenylation

Splicing proceeds in what two steps?

1. cleavage at 5' splice site and joining of 5' end of intron to A within intron (intron forms a loop)

2. cleavage at 3' splice site and simultaneous ligation of exons excises intron loop

What are three important sequence elements of pre-mRNAs?

1. 5' splice site

2. 3' splice site

3. branch point within intron

What are spliceosomes?

five types of small nuclear RNAs (snRNAs): U1, U2, U3, U4, U5, U6

snRNAs are complexed with 6-10 protein molecules to form ___

small nuclear ribonucleoprotein particles (snRNPs)

How are spliceosomes assembled?

1. binding of U1 snRNP to 5' splice site (SS)

2. base pairing between 5' SS sequence and 5' end of U1 snRNA

3. U2 snRNP binds to branch point

4. other snRNPs join complex and act together to form intron loop and maintain association of 5' and 3' exons so they can be ligated

5. excision of the intron

What is RNA editing?

alters the protein-coding sequences of mRNAs

What is pre-mRNA?

90% of the sequence are introns, degraded in the nucleus after splicing, processed mRNAs are protected by capping and polyadenylation

How are eukaryote mRNAs degraded?

initiated by shortening of poly-A tails

How are eukaryotic mRNAs regulated?

extracellular signals such as growth factors and hormones

degradation regulated by siRNA and miRNA