Chapter Eleven Chapter Eleven Transcription of the Genetic Code: - - PDF document

Mary K. Campbell Shawn O. Farrell

• Chapter Eleven

Paul D. Adams • University of Arkansas

Chapter Eleven Transcription of the Genetic Code: The Biosynthesis of RNA

1

Transcription

2

3

Transcription in Prokaryotes

• E. coli RNA Polymerase:
• molecular weight about 500,000
• four different types of subunits: α, β , β’, and σ
• the core enzyme

core enzyme is α2ββ’

• the holoenzyme

holoenzyme is α2ββ’σ

• the role of the σ subunit is recognition of the promoter

promoter locus locus; the 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 strand strand; the other is called the coding (or nontemplate) strand

• r sense strand

sense strand

• the holoenzyme binds to and transcribes only the template

strand

4

The Basics of Transcription

5

Coding strand

6

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 is first nucleotide of gene to be transcribed is

• Promoters-DNA sequence that provide direction for

RNA polymerase

7

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?

8

9

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

10

Initiation and Elongation in Transcription

11

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. ribonucleotides ribonucleotides

• Topoisomerases relax supercoils in front of and

behind transcription bubble

12

Chain Elongation (Cont’d)

13

Chain Termination

• Two types of termination mechanisms:
• intrinsic termination- controlled by specific

sequences, termination sites

• Termination sites characterized by two inverted

repeats

14

Chain Termination (Cont’d)

• Other type of termination involves rho (ρ) protein
• Rho-dependent termination sequences cause hairpin

loop to form

15

Transcription Regulation in Prokaryotes

• In prokaryotes, transcription regulated by:
• alternative σ factors
• enhancers
• operons
• transcription attenuation
• transcription attenuation
• Alternative σ

σ σ 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.

16

Control by Different σ Subunits

17

Operon

• Operon

Operon: 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

β β β β β β β β-Galactosidase Galactosidase, 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

18

How Does Repression Work

• Repressor protein

made by lacI gene forms tetramer when it is translated

• Repressor protein then

binds to operator binds to operator portion of operon

• Operator and promoter

together are the control sites

19

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

20

Binding Sites On lac operon (Cont’d)

21

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

22

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

enhancers enhancers

• Bound by proteins called transcription factors

23

Elements of a Bacterial Promoter

24

Basic Control Mechanisms in Gene Control

• Control may be inducible or repressive, and these

may be negatively or positively controlled

25

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

26

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

27

Attenuation in the trp operon

• Pause structure

forms when ribosome passes

• ver Trp codons

when Trp levels are high high

• Ribosome stalls at

the Trp codon when trp levels are low and antiterminator loop forms

28

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

29

RNA Polymerase II

• Most studied on the polymerases
• Consists of 12 subunits
• RPB- RNA Polymerase B

30

How does Pol II Recognize the Correct DNA?

• Four elements of the Pol II promoter allow for this

phenomenon

31

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

32

General Transcription Initiation Factors

33

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

34

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

35

Gene Regulation

• Enhancers and silencers- regulatory sequences

that augment or diminish transcription, respectively

• DNA looping brings enhancers into contact with

transcription factors and polymerase

36

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

37

Activation of transcription Via CREB and CBP

• Unphosphorylated

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

• Phosphorylation of

CREB causes binding

• f CREB to CBP
• Complex with basal

complex (RNA polymerase and GTFs) activates transcription

CRE – cAMP-response element CREB – cAMP-response element binding protein CBP – CREB-binding protein PKA – cAMP dependent protein kinase (protein kinase A)

38

Response Elements

39

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 translocation translation, protein stabilization, protein translocation

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

Interfering RNA (siRNA)

40

SiRNAs are formed in away similar miRNA

41

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

42

Helix-Turn-Helix Motif

Hydrogen bonding between amino acids and DNA

43

Zinc Finger Motif

• Motif contains 2

cysteines and 2 His 12 amino acids later

• Zinc binds to the

repeats repeats

44

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

45

Helical Wheel Structure of Leucine Zipper

46

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 acids domains, 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

47

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 mRNA

• Modifications
• trimming of leader and trailer sequences
• addition of terminal sequences (after transcription)
• modification of the structure of specific bases (particularly

in tRNA)

48

Posttranscriptional Modification of tRNA Precursor

49

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 modification all take place

• methylation and

substitution of sulfur for

• xygen are the two most

usual types of base modification

50

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

51

Modification of mRNA

• Includes the capping
• f the 5’ end with an

N-methylated guanine that is bonded to the next residue by a 5’ -> 5’ triphosphate.

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

52

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 exons

exons

• Intervening DNA sequences that are not expressed

are called introns introns

• These genes are often referred to as

These genes are often referred to as split genes split genes

53

Organization of Split Genes in Eukaryotes

54

The Splicing Reaction

• Exons are

separated by intervening intron

• When the exons

are spliced are spliced together,a lariat forms in the intron

55

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

56

Self-splicing of pre-rRNA

57