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JIB 322 Molecular Biology Gene transcription & RNA modification (chapter 12) 1

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JIB 322 – Molecular Biology

Gene transcription & RNA modification (chapter 12)

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DNA makes RNA makes protein

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Transcription• Two fundamental concepts:• 1. DNA sequences provide the information for

gene transcription (structural + regulatory)• 2. Interactions between proteins with the DNA

sequences to regulate the transcription process.

• Gene transcription actively studied in the areas of developmental biology, cancer biology, and biotechnology.

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• At the molecular level gene expression is the process by which the information in a gene is used to make a functional product, often a protein.

• The transcriptional unit contains base sequences that have different functions during transcription

• The promoter provides the site to begin transcription.

• The terminator signals the end of transcription.• The RNA transcript is complementary to the

template strand.• For structural genes that encode proteins, the non-

template strand is called the coding, or sense, strand.

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• Regulatory sequences are involved in the regulation of gene expression - act as binding sites for transcription factor proteins.

• In bacteria, ribosome-binding sites (rbs) provide a site for ribosome attachment to begin translation of mRNA .

• During translation, for both eukaryotes and prokaryotes, the mRNA is read as a series of three bases called a codon.

• The first codon is called the start codon (AUG/ Met)• The stop codon (UAA, UAG, UGA) signals an end to

translation.

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Transcription• Occurs in 3 stages i.e. initiation, elongation (or

synthesis), and termination. All three stages involve protein-DNA interactions.

• DNA in the form of a double-helix is called a closed promoter complex. These strands must be separated for transcription to begin.

• The RNA polymerase forms an open promoter complex (also called an open complex), separating the DNA into template and non-template strands.

• Termination signals the RNA polymerase to dissociate with the DNA.

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Transcription in Bacteria• During the 1950s and 1960s researchers studied gene expression

in bacteria and bacteriophages. E. coli is the model organism for many of the studies of bacterial transcription.

• • Promoters Initiate Transcription• The promoter directs the exact location for the initiation of RNA

transcription.• Promoters are located upstream of the start site.• The numbering of bases corresponds to the location of the

transcriptional start site• Sequence elements in promoter that are crucial to its function.• In E. coli, the -10 region and -35 region are important.• The -35 region is also called the Pribnow box.- these regions typically have a consensus sequence of bases

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Variation in promoter consensus sequences

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• RNA polymerase catalyzes RNA synthesis (elongation stage).

• The core enzyme composed of α2ββ’ subunits.• The addition of the sigma () factor creates a holoenzyme.• Roles of the RNA polymerase subunits:• The alpha (α) subunits are involved in polymerase

assembly and binding to the DNA.• The beta (β) subunits are involved in DNA binding

and catalytic processes.• The sigma () factor recognizes the promoter.• Proteins that influence the function of the RNA

polymerase are called transcription factors.

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• The RNA holoenzyme binds loosely to the DNA and moves along the strands until the sigma factor recognizes the -10 and -35 regions of the promoter.

• An internal region of the sigma factor, with a helix-turn-helix structure, then promotes tighter binding to the DNA - involving an interaction with the major groove of the DNA.

• Once bound to the closed promoter complex, unwinding of the DNA begins at the -10 region.

• This occurs in the TATAAT bases of the -10 region. This is due to the fewer hydrogen bonds between A and T.

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Helix turn helix interaction with major groove of DNA

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Elongation of transcription• The RNA transcript is made during the elongation stage. The

RNA polymerase moves along the DNA, causing it to unwind.• The noncoding strand is used as template.• The coding strand has the same sequence as the RNA transcript,

except that U is substituted for T in the DNA.• The open complex that is formed by the moving RNA

polymerase is 17 bp long.• The RNA polymerase moves at a rate of 43 bp per second. The

RNA polymerase connects bases according to the AT/GC rule.• The RNA polymerase connects nucleotides in a 5’ to 3’ direction

by creating a bond between the 5’ phosphate group of one nucleotide and the 3’ –OH group of the second nucleotide.

• Multiple transcript events can occur in close proximity, and both strands of DNA can contain genes

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Transcription of three different genes on same chromosome

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Termination of Transcription

• In E coli, there are two different mechanisms of termination, called rho-dependent termination and rho-independent termination.

• Rho-dependent has two components, the rho () protein and a binding site for rho, plus the formation of a stem-loop in the RNA transcript. The rho protein acts as a helicase, which separates the DNA-RNA hybrid regions.

• The rho protein attaches to the RNA transcript at the rut site (rho utilization site).

• A sequence of bases in the DNA encodes for a stem-loop structure in the RNA, causing a pause in transcription. During this pause the rho protein catches up to the open complex and disrupts the DNA-RNA hybrid.

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dependent termination

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• In rho-independent termination, the RNA forms a uracil rich stem-loop structure. The stem-loop region causes a pause in transcription, and the pause is stabilized by NusA protein.

• The uracil-rich region in the open complex is unstable, causing the RNA transcript to dissociate from the DNA, terminating transcription. This is known as intrinsic termination.

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independent termination (intrinsic termination)

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Transcription in Eukaryotes• There are three RNA polymerases in eukaryotes.• RNA polymerase I transcribes ribosomal

RNA (rRNA) genes except 5S rRNA gene.• RNA polymerase II transcribes all structural

genes (mRNA).• RNA polymerase III transcribes transfer RNA

(tRNA) and the 5S rRNA genes.• There is structural similarity between the bacterial

RNA polymerases and eukaryotic RNA polymerases

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similarity between the bacterial and eukaryotic RNA polymerases

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Eukaryotic Structural Genes• The eukaryotic promoter contains a transcriptional

start site, a TATA box, and regulatory elements .• The core promoter is short and consists of a

TATAAAA sequence called the TATA box.• The TATA box is usually located at -25 bp and

determines the start site for transcription.• The core promoter can produce a low level of

transcription on its own @ basal transcription.• Regulatory elements influence the ability of the

RNA polymerase to recognize the core promoter.

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Structure of typical eukaryotic promoter

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Transcription regulation in eukaryotes• Enhancers act as activating sequences.• Silencers repress transcription.• Regulatory transcription factors are proteins that bind

to these regulatory regions.• Regulatory elements may be located some distance

from the gene being transcribed.• DNA sequences such as the TATA box, enhancers, and

silencers are called cis-acting elements since they influence a nearby gene.

• Trans-acting factors are produced by regulatory genes that are located some distance from the transcribed gene.

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Transcription Initiation in Eukaryotes• Three categories of proteins are needed for RNA

transcription of structural genes in eukaryotes. These are RNA polymerase II, general transcription factors, and mediator.

• The combination of the RNA polymerase and the general transcription factors is called a basal transcription apparatus.

• Mediator serves as the interface between the RNA polymerase and regulatory signals. It acts as the switch between transcriptional initiation and elongation in eukaryotes.

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Table 12.2

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Action of TF in eukaryotes leading to formation of open complex

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Chromatin and Gene Transcription• Compaction of DNA can be an obstacle to transcription.• The RNA polymerase and general transcription factors are not

able to interact with DNA packaged within a nucleosome.• Chromatin structure may be altered by :• Histone acetyl-transferases loosen the structure of the

nucleosome• Histone deacetylases restore a tighter interaction

between the histones and DNA.• ATP-dependent chromatin remodeling uses energy (ATP)

to alter the structure of the nucleosome by moving the nucleosomes from one location to another. The complexes that perform this reaction belong to a class of molecules called the SWI/SWF family, prevalent in eukaryotes.

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RNA Modification• The one-to-one correspondence of sequences in the

DNA coding strand and the sequence of the polypeptide is called colinearity. This occurs only in prokaryotic organisms.

• Eukaryotic organisms contain coding sequences (exons) and intervening sequences (introns).

• The entire gene sequence is synthesized during transcription, and then the exons are spliced together by a process called RNA splicing.

• Other RNA modifications also occur (Table 12.3, p.310).

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Cleavage of Large Transcripts• For nonstructural genes, the RNA transcript is

edited into smaller functional RNA molecules.• Examples are rRNA in mammals and tRNA• For tRNA editing, endonucleases act upon the

tRNA.• Ribozymes are RNA molecules that have

enzymatic activity.

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tRNA editing

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Phillip Leder used RNA-DNA hybridization to indicate the presence of introns in globin gene by the

existence of double stranded R loops in the hybrid

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Splicing Mechanisms• Splicing removes intron RNA and connects exon RNA by a

phosphodiester bond.• Self-splicing introns consist of group I and group II introns.• Group I introns involve a guanosine within the

transcript (Figure 12.20a).• Group II introns involve an adenine nucleotide

(Figure 12.20b).• Proteins called maturases assist the self-splicing introns in

their functions.• In eukaryotes, structural genes start as long pre-mRNA,

which are also called heterogenous nuclear RNA (hnRNA).• pre-mRNA is spliced by a spliceosome (Figure 12.20c).

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Spliceosomes

• A spliceosome is a large complex that splices pre-mRNA. It contains subunits called snRNPs (small nuclear RNA and a set of proteins).

• snRNPs allow for precise recognition of intron-exon boundaries.

• Intron-exon boundaries consist of highly conserved sequences (Figure 12.21).

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A spliceosome is a large complex that splices pre-mRNA. It contains subunits called snRNPs (small nuclear RNA and a set of proteins).

snRNPs allow for precise recognition of intron-exon boundaries.

Intron-exon boundaries consist of highly conserved sequences

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The molecular mechanism of spliceosome

The chemical basis of spliceosome activity is not completely understood, but it is believed that the molecule may act as a ribozyme

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Most mature mRNAs have a 7-methylguanosine cap at the 5’ end. This is called capping.

a. Allows for the exit of certain RNAs from the nucleus.b. The cap is involved in the early stages of translation.c. The cap may also be involved with the splicing of introns at the 5’ end.

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Most mature mRNAs have a string of adenine nucleotides, called a polyA tail, at the 3’ end. This requires a polyadenylation sequence in the transcript . Important in the stability of the mRNA and in translation.