BIOL 3301 Chapter 12 Gene Transcription and RNA Modification

gene

• at molecular level, a ? is a. segment of DNA used to make a functional product, like RNA or a polypeptide

transcription

• first step in gene expression

• note: not every gene in your genome is turned on/expressed in all cells all the time

• your liver cells aren’t the same as kidney cells bc they have different proteins encoded in your genome.

transcription

• act or process of making a copy

• in genetics, copying of a DNA sequence into an RNA sequence

• DNA is structure is NOT altered from this process

  • it can continue to store information

protein coding/structural genes

• code the amino acid sequence of a polypeptide

  • transcription of protein coding gene produces mRNA

  • mRNA base sequence determines the amino acid sequence of a polypeptide during translation

  • 1 or more polypeptides constitute a protein

  • synthesis of functional proteins determines an organism’s traits

mRNA base sequence

• determines the amino acid sequence of a polypeptide during translation

DNA replication

• makes DNA copies that are transmitted from cell to cell and from parent to offspring

chromosomal DNA

• stores information in units called genes

transcription

• produces an RNA copy of gene

messenger RNA

• a temporary copy of a gene that contains information to make a polypeptide

translation

• produces a polypeptide using the information in mRNA

polypeptide

• becomes part of a functional protein that contributes to an organism’s traits

central dogma of genetics

• DNA replication

  • transmitted from cell to cell and from parent to offspring

• chromosomal DNA

  • stores info in genes

• transcription

  • produce RNA copy of gene

• messenger RNA

  • temporary copy of gene w/ info to make polypeptide

• translation

  • produce polypeptide using mRNA info

• polypeptide

  • becomes part of functional protein that contributes to an organism’s traits

central dogma of genetics

• DNA → RNA (transcription) → Polypeptide (translation) → Protein (functional role)

• The DNA stores the information.

• The information is copied into mRNA through transcription.

• The mRNA is used to make a polypeptide through translation.

• The polypeptide folds into a protein that performs a function, contributing to the organism's traits.

transcription

• DNA base sequences define the beginning and end of a gene and regulate the level of RNA synthesis.

• The DNA sequence:

  • Specifies where transcription begins (promoter).

  • Specifies where transcription ends (terminator).

  • Determines what should be made (e.g., mRNA, tRNA, etc.).

  • Regulates how much RNA is made

    • enhancers: increase RNA production

    • silencers: decrease RNA production

• Sequences in DNA signal to proteins that:

  • Recognize and read the sequences.

  • Follow the instructions from the gene information to produce proteins or other functional molecules (like tRNA).

• Proteins (RNA polymerase and transcription factors) must recognize and act on DNA for transcription to occur.

  • transcription factors: proteins that recognize specific DNA sequences and control transcription rate

gene expression

• overall process by which the information within a gene is used to produce a functional product which can, in concert with environmental factors, determine a trait

• (A gene's information is first transcribed into RNA, which is then translated into a protein. This protein, functioning within a specific environment, ultimately contributes to the expression of a particular trait)

transcription

• in a DNA molecule, the DNA sequence itself says where the beginning and end are, how to start and stop, and what should be made from that sequence and also how much

• those sequences are signals to the proteins that can “recognize” those sequences and actually do the work of “reading” the information in a gene and following the instructions in that information to make more proteins, or other functional molecules (like tRNAs)

sequence organization to form a bacterial gene that codes an mRNA transcription

• DNA

  • regulatory element

    • site for binding of regulatory proteins

      • regulatory proteins influence transcription rate

    • found in variety of locations

  • promotor

    • RNA polymerase binding site

    • signals beginning of transcription

  • terminator

    • signals end of transcription

• mRNA

  • ribosome binding site

    • site for ribosome binding

    • translation begins near this site in mRNA
      in eukaryotes, ribosome scans mRNA for start codon

  • start codon

    • specifies 1st amino acid in a polypeptide sequence

      • formylmethionine (bacteria)

      • methionine (eukaryotes)

  • codons

    • 3 nucleotide sequences within mRNA that specify particular amino acids

    • codon sequence within mRNA determines the sequence of amino acids within a polypeptide

  • stop codon

    • specifies the end of a polypeptide synthesis

  • bacterial mRNA may be polycistronic (codes 2 or more polypeptides)

regulatory elements

• specific DNA sequences that serve as binding sites for regulatory transcription factor proteins

  • these regulatory proteins (when bound) influence transcription rate

• found in a variety of locations (upstream, downsream of gene or within introns)

• DNA

promotor

• site for RNA polymerase binding

• signals beginning of transcription

  • dictates where RNA polymerase should start synthesizing RNA

• DNA sequence

terminator

• signals end of transcription

  • when RNA polymerase encounters ?, RNA polymerase detaches from DNA, releasing newly synthesized RNA molecule

• DNA sequence

ribosome binding site

• site for ribosome binding to mRNA in bacteria

• translation begins near this site in the mRNA

  • in eukaryotes, the ribosome binds to a 7-methyguanosine cap in mRNA and ribosome scans the RNA for a start codon

• mRNA

codons

• 3 nucleotide sequences within the mRNA that specify particular amino acids

• ? sequence within mRNA determines the sequence of amino acids within a polypeptide chain during translation

• mRNA

start codon

• specifies the 1st amino acid in a polypeptide sequence (initiate translation)

• formylmethionine (bacteria)

• methionine (eukaryotes)

• mRNA (AUG)

stop codon

• specifies the end of polypeptide synthesis (translation)

• mRNA

polycistronic

• bacteria mRNA may code 2 or more polypeptides

template/noncoding/antsense strand

• DNA strand that is actually transcribed (used as template) for RNA synthesis during transcription

  • RNA transcript is complementary to this sequence

coding/sense/nontemplate strand

• DNA strand not used as template during transcription (opposite to template strand)

• base sequence in RNA is identical to the ? strand of DNA, except for the substitution of uracil in RNA for thymine in DNA

transcription factors

• recognize the promotor and regulatory elements to control transcription rate

  • bind to enhancers (DNA), silencers (DNA)

• proteins that bind to specific DNA sequences

mRNA

• ? sequences like ribosomal binding site and codons direct translation

  • ribosomal binding site in bacteria (or 5’ cap in eukaryotes) position ribosome correctly on mRNA

  • codons specify amino acids to be incorporated into growing polypeptide chain

transcription stages

• 3 stages

  • Initiation

    • RNA polymerase binds promotor region on DNA→ initiate unwinding of DNA double helix

  • Elongation

    • RNA polymerase moves along DNA template strand, synthesizing complementary RNA molecule in 5’ to 3’ direction

  • Termination

    • RNA polymerase encounters terminator DNA sequence→ release RNA transcript

• involve protein-DNA interactions

  • proteins like RNA polymerase and various transcription factors interact with DNA sequences (promotors, terminators, regulatory elements) to regulate gene expression

initiation

• promotor functions as a recognition site for transcription factors

• transcription factors enable RNA polymerase to bind to the promotor region of DNA

• after binding, the DNA is denatured into an open transcription bubble (double helix→ open complex transcription bubble)

• transcription stage

elongation/synthesis of RNA transcript

• RNA polymerase slides 5’ to 3’ along DNA template strand in an open complex to synthesize complementary RNA

  • continuous addition of ribonucleotides to growing mRNA transcript

    • A with U

    • T with A

    • C with G

    • G with C

• transcription stage

termination

• a terminator DNA sequence is reached that causes RNA polymerase and the end RNA transcript to dissociate from the DNA template

• transcription stage

bacteria

• our molecular understanding of gene transcription came from early studies involving ? and ?phages

• much of our knowledge coms from studies of E. coli

promoters

• DNA sequences that “promote” gene expression

• direct the exact location for transcription initiation

• typically immediately upstream of site where transcription of a gene actually begins

• bases in a ? sequence are numbered in relation to the transcriptional start site (allow precise ID of regulatory elements)

  • no zero nucleotide in this numbering system

  • negative numbers ID bases preceding beginning of transcription (to the left)

  • vary at the -35 and -10 sequences

    • most common is consensus sequence

      • consensus sequence likely to result in high level of transcription

    • sequences that deviate from consensus sequence typically result in lower levels of transcription

template strand

• the one “read” by RNA polymerase to synthesize a complementary RNA molecule

coding strand

• the one that has the same sequence as the RNA transcript (except it has Thymidines rather than Uracils)

  • the DNA strand with same sequence as mRNA transcript (but with T instead of U)

  • not used as a template for RNA synthesis

consensus sequence

• most common promotor sequence at the -35 and -10 sequences

• likely to result in a high level of transcription

  • if sequence deviates from the ? sequence it typically results in lower levels of transcription

• DNA

initiation of bacterial transcription

• RNA polymerase catalyzes RNA synthesis

• RNA polymerase holoenzyme binds loosely to the DNA and scans along the DNA until it encounters a promotor

  • in E. coli, RNA polymerase holoenzyme is composed of

    • core enzyme (5 subunits) responsible for transcription

    • sigma factor (1 subunit) protein helping RNA polymerase locate the promotor

  • these subunits play distinct functional roles

• when RNA polymerase encounter a promotor, sigma factor recognizes both the -35 and -10 promotor sequences

  • a region within the sigma factor, containing a helix-turn-helix structure, facilitates tighter binding to the DNA (this binding forms the closed complex)

• binding of RNA polymerase to promotor forms closed complex

• then the TATAAT box in the -10 sequence is unwound, forming the open complex, and a short RNA strand is made

  • A-T bonds more easily separated, aiding in unwinding

  • sigma factor is released (after synthesizing the short RNA strand within the open complex)

    • core enzyme is able to proceed along the DNA marking the end of initiation and beginning of elongation where core enzyme now slides down DNA to synthesize an RNA strand

• bacterial transcription

RNA polymerase

• enzyme that catalyzes the synthesis of RNA in bacterial initation of transcription

• bacterial transcription

core enzyme

• part of RNA polymerase holoenzyme that does the transcription

• 5 subunits

• bacterial transcription

sigma factor

• protein that is a part of RNA polymerase holoenzyme, helps RNA polymerase find the promotor

• 1 subunit

• recognizes the -35 and -10 promotor DNA sequences as RNA polymerase holoenzyme scans the DNA for a promotor

  • contains a region with a helix turn helix structure that helps the RNA polymerase bind tighter to the DNA promotor→ closed complex formation

  • ? is released when the short RNA strand is synthesized within the open complex

• bacterial transcription

RNA polymerase holoenzyme

• responsible for bacterial transcription, composed of

  • core enzyme

    • 5 subunits

    • catalyzes RNA synthesis (does the transcription)

  • sigma factor

    • 1 subunit

    • protein that helps RNA polymerase find the promotor

• loosely binds to DNA and scans for a promoter. Upon encountering a promoter, the sigma factor facilitates binding, leading to the formation of a closed complex, which subsequently transitions into an open complex

elongation in bacterial transcription

• RNA transcript is synthesized

  • RNA polymerase slides along the DNA in a 3’ to 5’ direction, creating an open complex as it moves

  • RNA is synthesized in a 5’ to 3’ direction using nucleoside triphosphates as precursors

    • pyrophosphate is released

  • complementary rule is the same as the AT/GC rule except U is substituted for T in the RNA

• each gene uses one DNA strand as a template, but the template strand used is not always the same

  • DNA strand used as template for RNA synthesis is the template or antisense strand

    • the opposite DNA strand is the coding strand

      • has the same base sequence as the RNA transcript except that T in DNA corresponds to U in RNA

  • a promotor specifies the direction of transcription

    • with regard to adjacent genes along a chromosome, some promoters direct transcription in one direction and others direct transcription in the opposite direction

      • in any case, the template strand is read in the 3’ to 5’ direction (direction of transcription)

      • synthesis of RNA transcript occurs in a 5’ to 3’ direction

promoter

• specifies the direction of transcription

  • for adjacent genes along a chromosome, some direct transcription in one direction and others direct transcription in the opposite direction

• for transcription of multiple genes within a chromosome, the direction of transcription and the DNA strand used as a template can vary among genes

  • template strand is always read in the 3’ to 5’ direction (direction of transcription is 3’ to 5’)

    • RNA synthesis occurs in a 5’ to 3’ direction

• in the figure, Genes A and B are transcribed L to R (promotor to terminator)

  • template strand is the bottom strand (bc direction of transcription is 3’ to 5’/read template strand from 3’ to 5’)

  • Gene A and B RNA is synthesized 5’ to 3’ to the right

• Gene C is transcribed R to L (promotor to terminator))

  • template strand is top strand (bc direction of transcription is 3’ to 5’/read template strand from 3’ to 5’)

  • Gene C RNA is synthesized 5’ to 3’ to the left

• always remember

  • direction of transcription/template strand reading is always 3’ to 5’

  • RNA transcript is always synthesized 5’ to 3’

elongation in bacterial transcription

• RNA pol slides along DNA 3’ to 5’, creating an open complex as it moves

• DNA template strand used to make a complementary copy of RNA, resulting in a short RNA DNA hybrid

• as RNA pol moves along template strand 3’ to 5’, RNA is synthesized 5’ to 3’ using nucleoside triphosphates as precursors

  • pyrophosphate is released

• complementarity rule is similar to AT/GC rule except U is substituted for T in RNA

termination of bacterial transcription

• end of RNA synthesis

• occurs when short RNA-DNA hybrid of the open complex is forced to separate

  • this releases RNA polymerase and newly made RNA

• E. coli has 2 diff mechanisms

  • rho-dependent

    • requires protein ρ (rho)

  • rho independent (intrinsic) termination

    • does not require ρ (rho) protein

    • facilitated by 2 sequences in RNA

      • stem loop structure upstream of uracil rich sequence

        • causes RNA polymerase to pause synthesis of RNA

      • uracil rich sequence at 3’ end of RNA

        • while RNA polymerase pauses, weakly bound U rich sequence can’t hold RNA-DNA hybrid together (leads to dissociation of the RNA pol and new RNA transcript from the DNA template)

rho independent intrinsic termination

• facilitated by 2 sequences in RNA

• stem loop structure upstream of uracil rich sequence

  • causes RNA polymerase to pause synthesis of RNA

• uracil rich sequence at 3’ end of RNA

  • while RNA polymerase pauses, weakly bound U rich sequence can’t hold RNA-DNA hybrid together (leads to dissociation of the RNA pol and new RNA transcript from the DNA template)

• mechanism for termination in E. coli bacteria

eukaryotic transcription

• more complex bc

  • larger, more complex cells (organelles)

    • added cellular complexity means more genes that code proteins are required (more protein-coding genes)

  • multicellularity adds another level of regulation

    • express genes only in the correct cells at the proper time

eukaryotic RNA polymerases

• nuclear DNA is transcribed by

  • RNA pol I

    • transcribes all rRNA genes (except for 5S rRNA)

  • RNA pol II

    • transcribes all protein-coding structural genes (basically synthesizes all mRNAs)

    • transcribes some snRNA genes needed for splicing

  • RNA pol III

    • transcribes all tRNA genes

    • transcribes the 5S rRNA gene

    • transcribes microRNA genes

• all 3 are very similar structurally and are composed of many subunits

• there is also remarkable similarity between bacterial RNA polymerase and its eukaryotic counterparts

eukaryotic RNA pol I

• transcribes all rRNA genes (except for 5S rRNA)

eukaryotic RNA pol II

• transcribes all protein-coding structural genes (basically synthesizes all mRNAs)

• transcribes some snRNA genes needed for splicing

eukaryotic RNA pol III

• transcribes all tRNA genes

• transcribes the 5S rRNA gene

• transcribes microRNA genes

eukaryotic transcription initation

• protein-coding genes are influenced by core promotor and regulatory elements

  • core promotor

    • relatively short DNA sequence necessary for transcription to occur

    • consists of TATA box, transcriptional start site, and 1 or more downstream promoter elements DPEs

      • TATA box not always present, but is important for determining the precise start point for transcription (so if it is missing, the transcription start site becomes undefined and transcription may start at a variety of different locations)

    • core promoter by itself produces a low level of transcription (basal transcription)

  • regulatory elements

    • enhancers

      • DNA segments, usually 50 bp to 1000 bp

      • contain 1 or more regulatory elements

      • vary widely in their locations but are often found in the -50 to -100 region

    • regulatory transcription factors

      • proteins that affect RNA pol’s ability to recognize core promoter and begin transcription

      • bind to regulatory elements and influence transcription rate

        • activators- recognize enhancers and stimulate transcription rate

        • repressors- bind enhancers, inhibit transcription

• factors regulating gene transcription can be divided into 2 general types

  • cis acting elements

    • DNA sequences that exert their effect only over a particular gene

    • TATA box, enhancers containing regulatory elements

  • trans acting factors

    • proteins (general and regulatory transcription factors) that bind to cis-acting elements

• 3 protein categories are required for basal transcription to occur at the promotor

  • RNA polymerase II

  • 6 general transcription factors (GTFs)

    • bc RNA polymerase can’t find promoters on its own

    • proteins that assist RNA pol II bind to promoter and initiate transcription

  • mediator (protein complex)

• general transcription factors and RNA polymerase II assemble at the promoter of a gene, which often contains a TATA box

core promoter

• relatively short DNA sequence necessary for transcription to occur

• consists of TATA box, transcriptional start site, and 1 or more downstream promoter elements DPEs

  • TATA box not always present, but is important for determining the precise start point for transcription (so if it is missing, the transcription start site becomes undefined and transcription may start at a variety of different locations)

• ? by itself produces a low level of transcription (basal transcription)

• eukaryotic transcription initiation

TATA box

• DNA sequence found within eukaryotic core promoters that determines transcription starting site

• important for determining the precise start point for transcription

• if it is missing, the transcription start site becomes undefined and transcription may start at a variety of different locations

• eukaryotic transcription initiation

basal transcription

• in eukaryotes, a low level of transcription produced by the core promoter (when alone)

• The binding of transcription factors to enhancer elements may increase transcription above this level.

• 3 protein categories required for this to occur at the promoter

  • RNA polymerase II

  • 6 general transcription factors (GTFs)

    • bc RNA polymerase can’t find promoters on its own

    • proteins that assist RNA pol II bind to promoter and initiate transcription

  • mediator (protein complex)

• general transcription factors and RNA polymerase II assemble at the promoter of a gene, which often contains a TATA box

enhancers

• DNA segments, usually 50 bp to 1000 bp

• contain 1 or more regulatory elements

• vary widely in their locations but are often found in the -50 to -100 region

• influences transcription in eukaryotes

regulatory transcription factors RTFs

• proteins that affect RNA pol’s ability to recognize core promoter and begin transcription

• bind to regulatory elements and influence transcription rate

  • activators- recognize enhancers and stimulate transcription rate

  • repressors- bind enhancers, inhibit transcription

• eukaryotic transcription initiation

activators

• regulatory proteins that recognize and bind to DNA sequences called enhancers

• stimulate transcription rate (otherwise most eukaryotic genes have low levels of basal transcription)

repressors

• proteins that bind to DNA sequences called enhancers

• inhibit eukaryotic transcription rate

cis acting elements

• DNA sequences that exert their effect only over a particular gene

• TATA box, enhancers containing regulatory elements

• regulate eukaryotic gene transcription

• “next to”

• may be located far away from core promoter, but always found within same chromosome as the genes they regulate.

trans acting factors

• proteins (general and regulatory transcription factors) that bind to cis-acting elements

• regulate eukaryotic gene transcription

• “across from”

• may be encoded by genes far away from genes they control, even on a diff chromosome

general transcription factors GTFs

• proteins necessary to initiate basal transcription at the core promoter

• proteins that assist RNA pol II bind to promoter and initiate transcription (bc RNA polymerase can’t find promoters on its own)

mediator

• large protein complex

• interacts with RNA pol II and various regulatory transcription factors

• depending on its interactions with RTFs, may stimulate or inhibit RNA pol II

basal transcription reqs

• 6 diff general transcription factors GTFs, mediator (protein complex, RNA pol II

eukaryotic transcript termination

• RNA pol II transcriptional termination

  • pre-mRNAs are modified by cleavage near 3’ end with subsequent attachment of a string of adenines to form a polyAtail

  • transcription terminates 500-2000 nucleotides downstream from polyA signal

  • 2 models for termination (unclear which/if correct)

    • allosteric

    • torpedo

eukaryotic transcription elongation

• RNA polymerase II: Moves along the DNA template strand, synthesizing RNA.

• 5' to 3' RNA synthesis: Builds the mRNA using ribonucleotide triphosphates.

• Maintaining the open complex: Unwinds DNA ahead of the polymerase and rewinds it behind.

• Proofreading: Corrects errors in the growing RNA strand.

• Modification of the mRNA: As RNA polymerase II transcribes, the growing pre-mRNA undergoes modifications, including 5' capping (shortly after initiation) and 3' polyadenylation (adding a poly-A tail). These modifications are crucial for mRNA stability and processing.

• Coordination with RNA processing: Couples transcription with modifications like splicing, ensuring the mRNA is ready for translation.

eukaryotic RNA modification

• RNA transcripts can be modified by

  • chopping

  • splicing

  • capping

  • tailing

• analysis of bacterial genes in the 1960s and 1970s revealed

  • DNA sequence in coding strand corresponds to nucleotide sequence in mRNA

  • codon sequence in mRNA provides instructions for amino acid sequence in the polypeptide

  • → termed the colinearity of gene expression

• analysis of eukaryotic protein-coding genes in the late 1970s revealed that they aren’t always colinear with their functional mRNAs

  • instead, coding sequences (exons) are often interrupted by intervening sequences (introns)

  • transcription produces pre-mRNA corresponding to entire gene sequence

    • introns are subsequently removed/excised

    • exons connected together or spliced

      • → RNA splicing

        • common genetic phenomenon in eukaryotes

        • (occasionally occurs in bacteria as well)

  • RNA transcripts can also be modified by

    • processing of rRNA and tRNA transcripts to smaller functional pieces (chopping)

    • 5’ capping

      • role in intron splicing, mRNA export from nucleus, and binding of mRNA to ribosome

    • 3’ polyA tailing of mRNA transcripts

      • RNA stability and translation in eukaryotes

• eukaryotic RNA modification

colinearity

• the direct correspondence between the sequence of codons in the DNA coding strand and the amino acid sequence of a polypeptide.

• in simple terms, the order of nucleotides in the DNA determines the order of amino acids in the protein

• but eukaryotic protein coding genes aren’t always ?

  • contain numerous introns interrupting exons

  • RNA splicing necessary to remove introns from the pre-mRNA transcription and produce functional mRNA molecule

• eukaryotic RNA modification

exons

• region of RNA molecule that remains after splicing has removed the introns

• in mRNA, contains the coding sequence of a polypeptide

• eukaryotic RNA modification

intron intervening sequences

• segment of RNA, specifically within a pre-mRNA transcript, that are removed during RNA splicing

• don’t code for protein and are excised to produce a continuous coding sequence in mature mRNA

• eukaryotic RNA modification

RNA splicing

• process by which introns, non-coding segments of RNA within a pre-mRNA transcript, are removed, and the remaining exons, coding segments, are covalently joined together to form a continuous mature mRNA molecule

• common in eukaryotes, occasionally occurs in bacteria

• RNA modification

chopping

• cleavage of a large rRNA or tRNA transcript into smaller pieces

• one or more of the smaller pieces becomes a functional RNA molecule

• transfer RNAs are also made as large precursors, and are cleaved at both the 5’ and 3’ ends to produce mature, functional tRNAs

• prokaryotic and eukaryotic rRNAs and tRNAs

• RNA modification

splicing

• involves cleavage and joining of RNA molecules

• RNA is cleaved at 2 sites, allowing an internal segment of RNA (intron) to be removed

• after intron is removed, the two ends of the RNA are joined together

• common among eukaryotic pre-mRNAs, occasionally with rRNAs, tRNAs, and a few bacterial RNAs

• RNA modification

capping

• attachment of a 7-methylguanosine cap to 5’ end of mRNA

• cap plays a role in splicing of introns, mRNA export from nucleus, mRNA binding to ribosome

• occurs on eukaryotic mRNAs

• RNA modification

tailing

• attachment of a string of adenine containing nucleotides to the 3’ end of mRNA at a site where the mRNA is cleaved

• important for RNA stability and translation in eukaryotes

• done on eukaryotic mRNAs and occasionally on bacterial RNAs

rRNA processing in eukaryotes

• in eukaryotes, rRNA gene is transcribed into a long 45S rRNA primary transcript

  • transcript is cleaved to produce 18S, 5.8S, and 28S rRNA molecules which become associated with protein subunits to form functional ribosomes

• occurs in nucleolus of cell nucleus

• chopping (rRNA) RNA modification

precursor tRNA processing

• 5’ End processing: RNaseP, an endonuclease, cleaves/cuts the precursor tRNA, creating the mature 5’ end

• 3’ End processing: RNase Z, another endonuclease, removes a segment from the 3’ end

• Intron Removal: If present, an intron is spliced out.  

• Base Modifications: Some bases within the tRNA are modified to other bases, such as methylguanosine, 2-isopentenyladenosine, pseudouridine, and 4-thiouridine (convert from G,A,U,C to the ones listed)

• note: RNase Z, splicing endonucleuase, tRNA ligase are proteins. RNase P is a complex btw an RNA molecule and a protein

• chopping (rRNA) RNA modification (eukaryotes)

splicing

• 3 mechanisms have been ID’ed

• all 3 cases involve

  • removal of the intron RNA from pre-mRNA transcript

    • intron RNA is defined by particular sequences within the intron and at the intron-exon boundaries

      • consensus sequences for pre-mRNA splicing in complex eukaryotes occur at the intron-exon boundaries and at a branch site within the intron itself

        • bases at these sites are highly conserved evolutionarily

  • covalent linkage of the exon RNA fragments to form mature mRNA molecule

  • eukaryotic RNA modification

capping

• most mature eukaryotic mRNAs have a 7 methyl guanosine covalently attached at their 5’ end

  • role of the cap

    • 7 methylguanosine cap structure is recognized by cap-binding proteins

      • cap binding proteins play roles in

        • movement of some RNAs out of the nucleus

        • early stages of translation

        • splicing of introns

• occurs as pre-mRNA is being synthesized by RNA pol II

  • usually when pre-mRNA transcript is only 20-25 bases long

• 3 step process

  • RNA 5’ triphosphatase removes one of the three phsophates

  • guanylyltransferase enzyme hydrolyzes GTP to GMP, which is attached to 5’ end

  • methyltransferase attaches a methyl group to the base guanine

• eukaryotic RNA modification

tailing/polyadenylation

• most mature mRNAs have a string of adenine nucleotides at their 3’ ends

  • polyA tail

• polyAtail is not coded in the gene sequence, but is added enzymatically after the gene is completely transcribed

  • an endonuclease cleaves pre-mRNA about 20 nucleotides downstream from 3’ end AAuAA sequence, making the pre-mRNA shorter at its 3’ end

  • poly A polymerase enzyme adds adenine nucleotides to the newly created 3’ end

• eukaryotic RNA modification (although some bacterial RNAs are also polyadenylated but is degraded at degradosome)

bacterial promoter

• consists of -35 and -10 sequences

eukaryotic promotor

• for protein coding genes, the core promoter often consists of a TATA box, a transcriptional start site, and downstream promoter elements

bacterial RNA polymerase

• a single RNA polymerase

eukaryotic RNA polymerase

• 3 types

• RNA pol II transcribes protein-coding genes

bacterial transcription initiation

• sigma factor is needed for promoter recognition

eukaryotic transcription initiation

• 6 general transcription factors and mediator assemble at core promoter

bacterial transcription elongation

• requires release of sigma factor

eukaryotic transcription elongation

• mediator controls the switch to the elongation phase via phosphorylation of the CTD domain

bacterial transcription termination

• rho dependent or rho independent

eukaryotic transcription termination

• according to the allosteric or torpedo model

bacterial splicing

• rare; self-splicing

eukaryotic splicing

• commonly occurs in protein-coding pre-mRNAs in complex ? via a spliceosome

• self-splicing is rare

• removal of introns in tRNAs is catalyzed by a splice endonuclease and tRNA ligase

7 methylguanosine cap

• occurs on nearly all mRNAs in eukaryotes

• does NOT occur in bacteria

• 5’ end

bacterial poly A tail

• sometimes added to the 3’ end of the mRNA

• promotes degradation (adding the poly A tail has opposite functional effect in bacteria vs eukaryotes)

eukaryotic poly A tail

• almost always added to the 3’ end of mRNAs

• promotes stability (adding the poly A tail has opposite functional effect in bacteria vs eukaryotes)

RNA editing

• occurs occasionally in eukaryotes

• not known to occur in bacteria