Paul D. Adams • University of Arkansas

Mary K. CampbellShawn O. Farrellhttp://academic.cengage.com/chemistry/campbell

Chapter ElevenTranscription of the Genetic Code:

The Biosynthesis of RNA

Transcription

• Overview of Transcription• synthesized on a DNA template, catalyzed by DNA-

dependent 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_____________ enzyme is 2’

• the _____________ _____________ is 2’

• the role of the subunit is recognition of the ____________________________; the 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 ____________ ____________ strandstrand; the other is called the coding (or nontemplate) strand or _____________ strand_____________ 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 factors• ___________ & ____________ exert control over

which genes are expressed by producing different -subunits that direct the RNA _____________ to different genes.

Control by Different 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 structure- binding between regions 1 and 2

• Terminator loop- binding between regions 3 and 4

• Antiterminator structure- Alternative 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 glutamine-rich 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 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

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