Mary K. CampbellShawn O. Farrellinternational.cengage.com/

Chapter Eleven

Paul D. Adams • University of Arkansas

Chapter ElevenTranscription of the Genetic Code: The

Biosynthesis of RNA

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Transcription

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Transcription in Prokaryotes

• E. coli RNA Polymerase:

• molecular weight about 500,000

• four different types of subunits: α, β , β’, and σ

• the core enzymecore enzyme is α2ββ’

• the holoenzymeholoenzyme is α2ββ’σ

• the role of the σ subunit is recognition of the promoterpromoter locuslocus; σthe σ subunit is released after transcription begins

• of the two DNA strands, the one that serves as the template for RNA synthesis is called the template strand or antisense antisense

strandstrand; the other is called the coding (or nontemplate) strand or sense strandsense strand

• the holoenzyme binds to and transcribes only the template strand

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The Basics of Transcription

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Coding strand

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Promoter Sequence

• Simplest of organisms contain a lot of DNA that is not transcribed

• RNA polymerase needs to know which strand is template strand, which part to transcribe, and where first nucleotide of gene to be transcribed isfirst nucleotide of gene to be transcribed is

• Promoters-DNA sequence that provide direction for RNA polymerase

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Promoter Sequence

� Promoters typically consist of 40 bp region on the 5'-side of the transcription start site

� Two consensus sequence elements:

• The "-35 region", with consensus TTGACA

• The Pribnow box near -10, with consensus TATAAT –this region is ideal for unwinding - why?

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Chain Initiation

• First phase of transcription is initiation

• Initiation begins when RNA polymerase binds to promoter and forms closed complex

• After this, DNA unwinds at promoter to form open

complex, which is required for chain initiation

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Initiation and Elongation in Transcription

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Chain Elongation

• After strands separated, transcription bubble of ~17 bp moves down the DNA sequence to be transcribed

• RNA polymerase catalyzes formation of phosphodiester bonds between the incorp. ribonucleotidesribonucleotides

• Topoisomerases relax supercoils in front of and behind transcription bubble

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Chain Elongation (Cont’d)

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Chain Termination

• Two types of termination mechanisms:

• intrinsic termination- controlled by specific sequences, termination sites

• Termination sites characterized by two inverted repeats

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Chain Termination (Cont’d)

• Other type of termination involves rho (ρ) protein

• Rho-dependent termination sequences cause hairpin loop to form

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Transcription Regulation in Prokaryotes

• In prokaryotes, transcription regulated by:

• alternative σ factors

• enhancers

• operons

• transcription attenuation• transcription attenuation

• Alternative σσσσ factors

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

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Control by Different σ Subunits

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Operon

•• OperonOperon:: a group of operator, promoter, and structural genes that codes for proteins

• the control sites, promoter, and operator genes are physically adjacent to the structural gene in the DNA

• the regulatory gene can be quite far from the operon

• operons are usually not transcribed all the time• operons are usually not transcribed all the time

�� ββββββββ--GalactosidaseGalactosidase,, an inducible protein

• coded for by a structural gene, lacZ

• structural gene lacY codes for lactose permease

• structural gene lacA codes for transacetylase

• expression of these three structural genes is controlled by the regulatory gene lacI that codes for a repressor

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How Does Repression Work

• Repressor protein made by lacI gene forms tetramer when it is translated

• Repressor protein then binds to operatorbinds to operator

portion of operon

• Operator and promoter together are the control sites

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Binding Sites On the lac operon

• Lac operon is induced when E. coli has lactose as the carbon source

• Lac protein synthesis repressed by glucose (catabolite repression)

• E. coli recognizes presence of glucose by promoter as it has 2 regions: RNA polymerase binding site, catabolite activator protein (CAP) binding site

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Binding Sites On lac operon (Cont’d)

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Catabolite Repression

• CAP forms complex with cAMP

• Complex binds at CAP site

• RNA polymerase binds at available binds at available binding site, and transcription occurs

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Enhancers

• Certain genes include sequences upstream of extended promoter region

• These genes for ribosomal production have 3 upstream sites, Fis sites

• Class of DNA sequences that do this are called enhancersenhancers

• Bound by proteins called transcription factors

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Elements of a Bacterial Promoter

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Basic Control Mechanisms in Gene Control

• Control may be inducible or repressive, and these may be negatively or positively controlled

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Control of the trp operon

• Trp operon codes for a leader sequence (trpL) and five polypeptides

• The five proteins make up 4 different enzymes that catalyze the multistep process that converts chorisimate to tryptophan

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Alternative 2˚ structures Can Form in trp Operon• These structures can

form in the leader sequence

• Pause structure-binding between regions 1 and 2

• Terminator loop-binding between regions 3 and 4

• Antiterminator structure- Alternative binding between regions 2 and 3

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Attenuation in the trp operon

• Pause structure forms when ribosome passes over Trp codons when Trp levels are highhigh

• Ribosome stalls at the Trp codon when trp levels are low and antiterminator loop forms

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Transcription in Eukaryotes

• Three RNA polymerases are known; each transcribes a different set of genes and recognizes a different set of promoters:

• RNA Polymerase I- found in the nucleolus and synthesizes precursors of most rRNAs

• RNA Polymerase II- found in the nucleoplasm • RNA Polymerase II- found in the nucleoplasm and synthesizes mRNA precursors

• RNA Polymerase III- found in the nucleoplasm and synthesizes tRNAs, other RNA molecules involved in mRNA processing and protein transport

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RNA Polymerase II

• Most studied on the polymerases

• Consists of 12 subunits

• RPB- RNA Polymerase B

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How does Pol II Recognize the Correct DNA?

• Four elements of the Pol II promoter allow for this phenomenon

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Initiation of Transcription

• Any protein regulator of transcription that is not itself a subunit of Pol II is a transcription factor

• Initiation begins by forming the preinitiation

complex

• Transcription control is based here

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General Transcription Initiation Factors

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Transcription Order of Events

• Less is known about eukaryotes than prokaryotes

• The phosphorylated Pol II synthesizes RNA and leaves the RNA and leaves the promoter region behind

• GTFs are left at the promoter or dissociate from Pol II

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Elongation and Termination

• Elongation is controlled by:

• pause sites, where RNA Pol will hesitate

• anti-termination, which proceeds past the normal termination point

• positive transcription elongation factor (P-TEF) and negative transcription elongation factor (N-TEF)negative transcription elongation factor (N-TEF)

• Termination

• begins by stopping RNA Pol; the eukaryotic consensus sequence for termination is AAUAAA

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Gene Regulation

• Enhancers and silencers- regulatory sequences that augment or diminish transcription, respectively

• DNA looping brings enhancers into contact with transcription factors and polymerase

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Eukaryotic Gene Regulation

• Response elements are enhancers that respond to certain metabolic factors

• heat shock element (HSE)

• glucocorticoid response element (GRE)

• metal response element (MRE)

• cyclic-AMP response element (CRE)

• Response elements all bind proteins (transcription factors) that are produced under certain cell conditions

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Activation of transcription Via CREB and CBP

• UnphosphorylatedCREB does not bind to CREB binding protein, and no transcription occurs

• Phosphorylation of CREB causes binding CREB causes binding of CREB to CBP

• Complex with basal complex (RNA polymerase and GTFs) activates transcription

CRE – cAMP-response elementCREB – cAMP-response element binding proteinCBP – CREB-binding proteinPKA – cAMP dependent protein kinase (protein kinase A)

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Response Elements

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Non-Coding RNAs

• As much as 98% of transcriptional output from human genomes may be comprised of non-coding RNAs (ncRNA)

• Linked to: regular transcription, gene silencing, replication, processing of RNA, RNA modification, translation, protein stabilization, protein translocationtranslation, protein stabilization, protein translocation

• Two main types: Micro RNA (miRNA), and Small Interfering RNA (siRNA)

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SiRNAs are formed in away similar miRNA

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Structural Motifs in DNA-Binding Proteins

• Most proteins that activate or inhibit RNA Pol II have two functional domains:

• DNA-binding domain

• transcription-activation domain

• DNA-Binding domains have • DNA-Binding domains have domains that are either:

• Helix-Turn-Helix (HTH)

• Zinc fingers

• Basic-region leucine zipper

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Helix-Turn-Helix Motif

Hydrogen bonding between amino acids and DNA

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Zinc Finger Motif

• Motif contains 2 cysteines and 2 His 12 amino acids later

• Zinc binds to the repeatsrepeats

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Basic Region Leucine Zipper Motif

• Many transcription factors contain this motif, such as CREB (Biochemical Connections, page 309)

• Half of the protein composed of basic region of conserved Lys, Arg, and His

• Half contains series of Leu

• Leu line up on one side, forming hydrophobic pocket

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Helical Wheel Structure of Leucine Zipper

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Transcription Activation Domains

• acidic domains- rich in Asp and Glu. Gal4 has domain of 49 amino acids, 11 are acidic

• glutamine-rich domains- Seen in several transcription factors. Sp1 has 2 glutamine-rich domains, one with 39 Glu in 143 amino acidsdomains, one with 39 Glu in 143 amino acids

• proline-rich domains- Seen in CTF-1 (an activator). It has 84 amino acid domain, of which 19 are Pro

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Post Transcriptional RNA Modification

• tRNA, rRNA, and mRNA are all modified after transcription to give the functional form

• the initial size of the RNA transcript is greater than the final size because of the leader sequences at the 5’ end and the trailer sequences at the 3’ end

• the types of processing in prokaryotes can differ greatly from that in eukaryotes, especially for mRNAfrom that in eukaryotes, especially for mRNA

• Modifications

• trimming of leader and trailer sequences

• addition of terminal sequences (after transcription)

• modification of the structure of specific bases (particularly

in tRNA)

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Posttranscriptional Modification of tRNA Precursor

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Modification of tRNA

• Transfer RNA- the precursor of several tRNAs is can be transcribed as one long polynucleotide sequence

• trimming, addition of terminal sequences, terminal sequences, and base modificationall take place

• methylation and substitution of sulfur for oxygen are the two most usual types of base modification

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Modification of rRNA

• Ribosomal RNA

• processing of rRNA is primarily a matter of methylation and trimming to the proper size

• in prokaryotes, 3 rRNAs in one intact ribosome

• in Eukaryotes, ribosomes have 80s, 60s, and 40s subunits

• base modification in both prokaryotes and eukaryotes is primarily by methylation

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Modification of mRNA

• Includes the capping of the 5’ end with an N-methylated guanine that is bonded to the next residue by a 5’ -> 5’ triphosphate.

• Also, 2’-O-methylation of terminal ribose(s)

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mRNA Modification

• A polyadenylate “tail” that is usually100-200 nucleotides long, is added to the 3’ end before the mRNA leaves the nucleus

• This tail protects the mRNA from nucleases and phosphatases

• Eukaryote genes frequently contain intervening base • Eukaryote genes frequently contain intervening base sequences that do not appear in the final mRNA of that gene product

• Expressed DNA sequences are called exonsexons

• Intervening DNA sequences that are not expressed are called intronsintrons

•• These genes are often referred to as These genes are often referred to as split genessplit genes

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Organization of Split Genes in Eukaryotes

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The Splicing Reaction

• Exons are separated by intervening intron

• When the exons are spliced are spliced together,a lariat

forms in the intron

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Ribozymes

• The first ribozymes discovered included those that catalyze their own self-splicing

• More recently, ribozymes have been discovered that are involved in protein synthesis

• Group I ribozymes

• require an external guanosine

• example: pre-rRNA of the protozoan Tetrahymena(next screen)

• Group II ribozymes

• display a lariat mechanism similar to mRNA splicing

• no requirement for an external nucleotide

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Self-splicing of pre-rRNA

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