Transcript Chapter 11 - Evangel University

Mary K. Campbell
Shawn O. Farrell
http://academic.cengage.com/chemistry/campbell
Chapter Eleven
Transcription of the Genetic Code:
The Biosynthesis of RNA
Paul D. Adams • University of Arkansas
Transcription
• Overview of Transcription
• synthesized on a DNA template, catalyzed by DNAdependent RNA polymerase
• ATP, GTP, CTP, and UTP are required, as is Mg2+
• no RNA primer is required
• the RNA chain is synthesized in the 5’ -> 3’ direction;
the nucleotide at the 5’ end of the chain retains its
triphosphate (ppp) group
• the DNA base sequence contains signals for initiation
and termination of RNA synthesis; the enzyme binds
to and moves along the DNA template in the 3’ -> 5’
direction
• the DNA template is unchanged
Transcription in Prokaryotes
• E. coli RNA Polymerase:
• molecular weight about ______________________
•
•
•
•
four different types of subunits: _____________________
the _____________ enzyme is 2’
the _____________ is 2’s
the role of the s subunit is recognition of the ______________;
the s subunit is released after ________________
• of the two DNA strands, the one that serves as the template for
RNA synthesis is called the template strand or ____________
strand; the other is called the coding (or nontemplate) strand
or _____________ strand
• the __________ binds to and transcribes only the ___________
The Basics of Transcription
Promoter Sequence
• Simplest of organisms contain a lot of DNA that is
__________________________
• RNA polymerase needs to know which strand is
_____________strand, which part to
_____________, and where first _____________
of gene to be transcribed is
• _____________ - DNA sequence that provide
direction for RNA polymerase
Promoter Sequence
Chain Initiation
• First phase of transcription is _____________
• _____________begins when RNA polymerase binds
to _____________and forms ________________
complex
• After this, DNA unwinds at _____________ to form
open complex, required for chain ____________
Initiation and Elongation in Transcription
Chain Elongation
• After strands separated, transcription
_____________ of ~17 bp moves down the DNA
sequence to be transcribed
• RNA polymerase catalyzes formation of
_____________bonds between the incorporated
_____________
• Topoisomerases _____________ _____________
in front of & behind __________ bubble __________
Chain Elongation (Cont’d)
Chain Termination
• Two types of termination mechanisms:
1. intrinsic termination- controlled by specific sequences called
_____________ _____________, characterized by two
_____________ _____________
Chain Termination (Cont’d)
2. Other type of termination involves rho () protein
-dependent termination sequences cause hairpin loop to form
Transcription Regulation in Prokaryotes
• In prokaryotes, transcription regulated by:
__________________________________
__________________________________
__________________________________
__________________________________
• Alternative s factors
• ___________ & ____________ exert control over
which genes are expressed by producing different
s-subunits that direct the RNA _____________ to
different genes.
Control by Different s Subunits
Enhancers
• Certain genes include sequences upstream of
____________________________________
• These genes for ribosomal production have _____
upstream sites, _________ sites
• Class of DNA sequences that do this are called
______________
• Bound by proteins called _________ _________
Elements of a Bacterial Promoter
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
• __________________, an inducible protein
• coded for by a structural gene, _________
• structural gene _________ codes for lactose
permease
• structural gene _________ codes for transacetylase
• expression of these 3 structural genes is controlled by
the regulatory gene ______ that codes for a repressor
How Does Repression Work
• Repressor protein
made by lacI gene
forms tetramer when
it is translated
• Repressor protein
then binds to
operator portion of
operon
• Operator and
promoter together
are the control sites
Binding Sites On the lac operon
• Lac operon is induced when E. coli has _________
as the carbon source
• Lac protein synthesis repressed by ____________
(catabolite repression)
• E. coli recognizes presence of glucose by promoter
as it has 2 regions: RNA polymerase binding site,
_________ _________ _________ (CAP) binding
site
Binding Sites On lac operon (Cont’d)
Catabolite Repression
• CAP forms
complex with
_________
• Complex binds at
CAP site
• RNA polymerase
binds at available
binding site, and
_____________
occurs
Basic Control Mechanisms in Gene Control
Control may be _________ or _________, and these
may be _________ or _________ controlled
Control of the trp operon
• Trp operon codes for a leader sequence (trpL) & 5
polypeptides
• The 5 proteins make up 4 different enzymes that catalyze the
multistep process that converts chorisimate to tryptophan
Alternative 2˚ structures can form in trp operon
• These structures can
form in the leader
sequence
• Pause structurebinding between
regions 1 and 2
• Terminator loopbinding between
regions 3 and 4
• Antiterminator
structureAlternative binding
between regions 2
and 3
Attenuation in the trp operon
• Pause structure
forms when
ribosome passes
over Trp codons
when Trp levels
are high
• Ribosome stalls at
the Trp codon
when trp levels are
low and
antiterminator loop
forms
Transcription in Eukaryotes
•
Three RNA polymerases are known; each
transcribes a different set of genes and recognizes
a _____________________________________:
• RNA Polymerase I- found in the _________ and
synthesizes precursors of most _________
• RNA Polymerase II- found in the _________ and
synthesizes _________ precursors
• RNA Polymerase III- found in the _________ and
synthesizes _________, other RNA molecules
involved in mRNA _________ and _________
_________
RNA Polymerase II
• Most studied
of the
polymerases
• Consists of
12 subunits
• ______ - RNA
Polymerase B
How does Pol II Recognize the Correct DNA?
4 elements of the Pol II promoter allow for this phenomenon
Initiation of Transcription
• Any protein regulator of transcription that is not itself
a subunit of Pol II is a _________ _________
• Initiation begins by forming a _________ _________
• Transcription control is based here
General Transcription Initiation Factors
Transcription Order of Events
• Less is known about
_________ than
_________
• The phosphorylated
Pol II synthesizes
RNA and leaves the
promoter region
behind
• GTFs are left at the
promoter or
dissociate from Pol II
Elongation and Termination
• Elongation is controlled by:
• _________ sites, where RNA Pol will hesitate
• _________, which proceeds past the normal
termination point
• _________ _________ _________ _________
(P-TEF) and _________ _________ _________
_________ (N-TEF)
• Termination
• begins by stopping RNA Pol; the _____________
consensus sequence for termination is ___________
Gene Regulation
• _________ & _________ - regulatory sequences
that augment or diminish transcription, respectively
• DNA _________ brings _________ into contact with
transcription _________ and ____________
Eukaryotic Gene Regulation
• _________ _________ are enhancers that respond
to certain metabolic factors
• _________ _________ _________(HSE)
• _________ _________ _________(GRE)
• _________ _________ _________(MRE)
• _________ _________ _________(CRE)
• Response elements all bind _________(transcription
factors) that are produced under certain cell
conditions
Response Elements
Activation of transcription Via CREB and CBP
• _________________
CREB does not bind
to CREB binding
protein, and no
transcription occurs
• _________________
of CREB causes
binding of CREB to
CBP
• Complex with basal
complex (RNA
polymerase and
GTFs) activates
_________________
Structural Motifs in DNA-Binding Proteins
• Most proteins that activate or
inhibit RNA Pol II have two
_________ _________ :
• DNA-binding domain
• transcription-activation domain
• DNA-Binding domains have
domains that are either:
• _________________________
• _________________________
• _________________________
Helix-Turn-Helix Motif
Hydrogen bonding between amino acids and DNA
Zinc Finger Motif
• Motif contains
2 cysteines and
2 His --12 amino
acids later
• Zn binds to the
repeats
Basic Region Leucine Zipper Motif
• Many transcription factors contain this motif,
such as CREB (Biochemical Connections,
page 315)
• 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
Helical Wheel Structure of Leucine Zipper
Transcription Activation Domains
• _________________________ domains - rich in
Asp and Glu. Gal4 has domain of 49 amino acids,
11 are acidic
• _________________________ domains - Seen in
several transcription factors. Sp1 has 2 glutaminerich domains, one with 39 Glu in 143 amino acids
• _________________________ domains - Seen in
CTF-1 (an activator). It has 84 amino acid domain,
of which 19 are Pro
Post Transcriptional RNA Modification
• ____________________________________ 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 _______________
• Modifications
• ____________________ of leader and trailer sequences
• addition of _____________ _________ (after transcription)
• modification of the structure of specific bases (particularly
in _______________)
Posttranscriptional Modification of tRNA Precursor
Modification of tRNA
• tRNA- the precursor of
several tRNAs is can be
transcribed as ________
polynucleotide sequence
• ____________________
____________________
____________________
all take place
• ____________________
& ________________ of
____________________
are the two most usual
types of base
modification
Modification of rRNA
• Ribosomal RNA
• processing of rRNA is primarily a matter of
________________________________________
• in _______________, 3 rRNAs in one intact ribosome
• in _______________, ribosomes have 80s, 60s, and
40s subunits
• base modification in both prokaryotes and eukaryotes
is primarily by _____________________
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)
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
sequences that do not appear in the final mRNA of
that gene product
• Expressed DNA sequences are called exons
• Intervening DNA sequences that are not expressed
are called introns
• These genes are often referred to as split genes
Organization of Split Genes in Eukaryotes
The Splicing Reaction
• Exons are separated
by intervening
_______________
• When the exons are
spliced together, a
_______________
forms in the intron
Ribozymes
• The first ribozymes discovered included those that
catalyze ______________________________
• More recently, ribozymes have been discovered that are
involved in _______________ _______________
• _______________ribozymes
• require an external guanosine
• example: pre-rRNA of the protozoan Tetrahymena
(next screen)
• _______________ribozymes
• display a ______ mechanism similar to mRNA splicing
• no requirement for an external nucleotide
Self-splicing of pre-rRNA