Chapter 11: Transcription

Transcription

Controls the expression of genes and the production of proteins

Primer, DNA template

In RNA synthesis, _____ is not needed, but a _____ is required

RNA polymerase

Enzyme that catalyzes the production of RNA on a DNA template

• Has a multisubunit structure - α, ω, β, β′, and σ

Core enzyme

Enzyme that lacks the sigma unit

• α2ωββ′

Holoenzyme

Enzyme that has all component parts, including coenzymes and all subunits

• α2ωββ′σ

Template strand

• Antisense or [—] strand

• Serves as a template for RNA synthesis

Coding strand

• Sense or [+] strand

• Contains the same sequence as the RNA that is synthesized from the template

Holoenzyme

Binds to specific DNA sequences and transcribes only the template strand

σ-subunit

Recognizes the promoter and is released after transcription begins

Promoter

Portion of DNA to which RNA polymerase binds at the start of transcription

• Contains a large amount of non-transcribed DNA

• Provide direction for RNA polymerase

  • Which strand is the template strand

  • Which part to transcribe

  • Where the first nucleotide of the gene to be transcribed is located

Transcription start site (TSS)

(+1 position) site where the first base to be incorporated into the RNA chain

Pribnow box

(-10 position) first promoter element located 10 bases upstream

-35 region

(-35 position) next promoter element located 35 bases upstream

Core promoter

Promoter
Area from -35 region to TSS

UP promoter

Promoter
Enhances the binding of RNA polymerase

Extended promoter

Promoter
Area from end of UP element to TSS

Chain initiation

• Part of transcription where RNA polymerase binds to DNA, the strands are separated, and the first nucleotide binds to its complement

• Begins when RNA polymerase binds to the promoter and forms closed complex

Closed complex

Complex that initially forms between RNA polymerase and DNA before transcription begins

Open complex

Form of the complex of RNA polymerase and DNA that occurs during transcription

Chain elongation

Occurs when the RNA polymerase is able to launch itself off the DNA promoter

• RNA polymerase scrunches the DNA into itself, causing torsional strain of the separated DNA strands

RNA polymerase

Catalyzes formation of phosphodiester bonds between the incorporated ribonucleotides

Topoisomerases

Relax the supercoils in front of and behind the transcription bubble

17

After strands are separated, transcription bubble of about _____ base pairs moves down the DNA sequence to be transcribed

Intrinsic termination

Not dependent on the rho protein and is controlled by termination sites

Termination sites

Areas in DNA that cause termination of transcription by generating hairpin loops and a zone of weak binding between DNA and RNA

Rho-dependent

_____ termination sequences cause a hairpin loop to form

Alternative σ factors

• Viruses and bacteria exert control over which genes are expressed by producing different σ-subunits that direct the RNA polymerase to different genes

• Examples

  • Virus that infects the bacteria Bacillus subtilis

  • Response of E. coli to heat shock

Enhancers

DNA sequences that bind to a transcription factor and increase the rate of transcription

Transcription factors

Proteins or other complexes that bind to DNA sequences and alter the basal level of transcription

upstream

Certain E. coli genes include sequences _____ of the extended promoter region

Fis sites

• Binding sites for the protein called Fis

• 3 upstream sites of the genes for ribosomal RNA production

Operons

• Group of operator, promoter, and structural genes

• Physically adjacent to the structural gene in the DNA

• Not transcribed all the time

• Regulated by induction
  
  

Structural gene

Directs the synthesis of a protein under the control of some regulatory gene

Induction

Triggering of the production of an enzyme by the presence of a specific inducer

Inducer

Molecule that turns on the transcription of a gene

lacI

Controls the expression of the three structural genes

β-galactosidase

Protein produced by lacZ

Lactose permease

Protein produced by lacY

Transacetylase

Protein produced by lacA

Regulatory gene

Directs the synthesis of a repressor protein

Repressor

Protein that binds to an operator gene, blocking the transcription and eventual translation of structural genes under the control of that operator

Lac repressor

Repressor protein made by the lacI gene forms a tetramer when translated

Operator

Where a repressor of protein synthesis binds

Control sites

(operator + promoter)

• Modulate the production of proteins whose amino acid sequence is specified by the structural genes under their control

induced

Lac operon is _____ when E. coli has lactose and no glucose available to it as a carbon source

Catabolite repression

When glucose and lactose are present, the cell does not make the lac proteins (repression of the synthesis of lac proteins by glucose)

cAMP

“hunger signal”

Promoter

Has RNA polymerase and catabolite activator protein (CAP) binding sites

Catabolite activator protein (CAP)

Binds to a promoter when complexed with cAMP, allowing RNA polymerase to bind to its entry site on the same promoter

Induction

• Negative control (Lactose operon)

• Positive control (catabolite repression)

Repression

• Negative control (trp operon)

• Positive control (inducer deletions are uninducible)

Repressor

_____ deletions are constitutive

Inducer

_____ deletions are uninducible

Autoregulation

The product of the trpR operon regulates its own production

Transcription attenuation

• A type of transcription control in which the transcription is controlled after it has begun via pausing and early release of incomplete RNA sequences

1·2 pause structure

Causes premature termination of transcription

3·4 terminator

Causes premature release of the RNA transcript

2·3 antiterminator

Allows transcription to continue

RNA Polymerase I

Found in the nucleolus and synthesizes precursors of most ribosomal RNAs

RNA Polymerase II

Found in the nucleoplasm and synthesizes mRNA precursors; also called RNA polymerase B (RPB)

RNA Polymerase III

Found in the nucleoplasm and synthesizes tRNAs, precursors of 5S ribosomal RNA, and other RNA molecules involved in mRNA processing and protein transport

Upstream elements, TATA box, TSS, downstream regulators

4 elements of Pol II promoters

Transcription

Upstream elements in _____: a portion of the sequences closer to the 3′ end than the gene to be transcribed, where the DNA is read from the 3′ to the 5′ end and the RNA is formed from the 5′ to the 3′ end

Translation

Upstream elements in _____: nearer to the 5′ end of the mRNA

TATA box

• Located 25 bases upstream of the TSS

• a DNA sequence that indicates where a genetic sequence can be read and decoded

Initiator element

Pol II promoter

Loosely conserved sequence surrounding the transcription start site in eukaryotic DNA

Downstream regulator

Pol II Promoter

More rare than upstream regulators

Transcription factor

Any protein regulator of transcription that is not itself a subunit of pol II is a _____

Preinitiation complex

Where most of the control of transcription occurs

• Phase where RNA polymerase and the general transcription factors bind to the DNA

General transcription factors (GTFs)

Six transcription factors that bind to DNA to initiate transcription

Pause sites

Where RNA pol will hesitate

Abortion

Premature termination

Antitermination

Proceeds past the normal termination point

stopping RNA polymerase

Termination begins by _____

AAUAAA

Eukaryotic consensus sequence for termination

Mediator

Giant protein complex that bridges the promoter and general transcription factors with remote silencers and enhancers

Enhancers and silencers

Regulatory sequences that augment or diminish transcription, respectively

DNA looping

Brings enhancers into contact with transcription factors and polymerase

Nucleosomes

Presence of _____ represses transcription

Chromatin remodeling complexes

Mediate ATP-dependent conformational changes in nucleosome structure that lead to transcription

Histone-modifying enzymes

Make covalent modifications to the histone core octamer

Chromatin

Transcription is regulated by modification of histone proteins in the _____

Response elements

Enhancers that respond to certain metabolic factors

Bind proteins (transcription factors) that are produced under certain cell conditions

• Heat-shock element (HSE)

• Glucocorticoid-response element (GRE)

• Metal-response element (MRE)

• Cyclic-AMP-response element

Cyclic-AMP-response element (CRE)

Important eukaryotic response element that is controlled by production of cAMP in the cell

Cyclic-AMP-response-element binding protein (CREB)

Binds to the CRE and activates transcription

CREB-binding protein (CBP)

Links the basal transcription machinery to CREB

Micro RNA (miRNA)

Affects gene expression and plays a role in growth and development

Small interfering RNA (siRNA)

Controls gene expression by selective suppression of genes

DNA-binding domain

Part of a transcription factor that binds to the DNA

• Helix–turn–helix (HTH)

• Zinc fingers

• Basic-region leucine zipper (bZIP)

Transcription-activation domain

Part of a transcription factor that interacts with other proteins and complexes rather than with the DNA directly

Helix-turn-helix (HTH)

• Proteins that bind to DNA consist of a segment of α-helix that fits into the major groove

• Contains a sequence of 20 amino acids that is relatively conserved in many different DNA-binding proteins

major

Proteins that recognize DNA with specific base sequences are more likely to bind to the _____ groove

Zinc fingers

• A group of proteins with structural features wrapped around a zinc ion, binding to specific amino acid residues

• Transcription factor of RNA polymerase III, TFIIIA, had nine repeating structures of 30 amino acids each

• Each repeat contained two cysteines and two histidines spaced after 12 amino acids

• Zinc ions bind to each of the repeats

Basic-region leucine zipper motif

• Present in many transcription factors, including CREB

• Half of the protein is composed of basic region, with conserved residues of lysine, arginine, and histidine

• Second half contains a series of leucines every seven residues

• With a seven-residue spacing, the leucines all line up on one side of an alpha-helix, forming a hydrophobic pocket

Acidic domains

• Rich in acidic amino acids

• Gal4 has a domain of 49 amino acids, 11 of which are acidic

Glutamine-rich domains

• Seen in several transcription factors

• Sp1 has two glutamine-rich domains, one with 39 glutamines in 143 amino acids

Proline-rich domains

• Seen in activator CTF-1

• CTF-1 has a domain of 84 amino acids, 19 of

  which are prolines

Trimming, Addition, Modification

Types of posttranscriptional RNA modification

Transfer RNA

• Trimming, addition of terminal sequences, and base modification take place in the transformation of the initial transcript to the mature tRNAs

• Usual types of base modification: methylation and substitution of sulfur for oxygen

Prokaryotes

Have three rRNAs in an intact ribosome, which has a sedimentation coefficient of 70S