Transcription & Translation L2

Histones have flexible tails that are post-translationally modified:

Ø Acetylation Ø Methylation Ø Phosphorylation Ø Ubiquitination Ø Poly-ADP-Ribosylation Ø Sumolation

Eukaryotic RNA processing: splicing steps

occurs in the nucleus 1. Splice donor site is cleaved 2. Attaches to the branch site to form a lariat or loop structure 3. The splice acceptor site is cleaved 4. The intron degrades, the two exons are ligated.

Transcription relies on

promoters RNA polymerase recruited to promoters through transcription factors that recognize the promoter region and organize a complex of proteins that bring in RNA polymerase to initiate transcrption

Eukaryotic Transcription: Termination RNA polymerase I (rRNA)

- pre-rRNA is transcribed and a complex of proteins (FACTOR DEPENDENT) is recruited to the termination site, leading to termination and subsequent cleavage. - There are several specific termination sequences upstream of a pause site, so factors move on RNA and catch up to the termination sequence (like the Rho-dependent mechanism in prokaryotes)

Transcription factors have 3 different domains

1. DNA binding domain 2. Signal sensing domain 3. Transcriptional activation domain

Eukaryotes have three distinct RNA polymerases:

1. RNA Polymerase I: 2. RNA Polymerase II: Synthesizes mRNA, and most snRNA and microRNA. 3. RNA Polymerase III: Synthesizes tRNA, 5S rRNA, and other small RNAs.

Modification of eukaryotic mRNA includes:

Capping Polyadenylation Splicing C-to-U editing

Eukaryotic mRNA Processing

Eukaryotic mRNA undergoes extensive processing in the nucleus, and some processing is required for transport to the cytoplasm::: as a result needs to be very stable Modifactions: Ø 5'-capping Ø 3'-poly(A) tail, -cap and tail protect the RNA Ø splicing to remove introns (mRNA) Ø cleavage, addition, ribose/base alterations (as in prokaryotes)

Readers

Proteins that recognize modified histones

Eukaryotic Transcription: Elongation

RNA poly reads template strand in the 3'->5' direction RNA is synthesized in the 5'->3' direction o In contrast to initiation, when only one RNAP molecule occupies a promoter at a time, elongation often involves multiple RNA Polymerases moving one after another along the same DNA duplex. o During elongation, the transcription machinery needs to move histones out of the way every time it encounters a nucleosome (ATP dependent enzymes can do this). o Elongation also involves a proofreading mechanism; replaces incorrectly incorporated bases (but does not function as well as DNA polymerase proofreading activity)

RNA polymerase I

Synthesizes rRNA (but not 5S rRNA) -unique in that it only transcribes only one set of genes (ribosomal RNA genes) -does not require a TATA-box in the promoter, rather relies on a distinct upstream control element (UCE) that recruits specialized proteins pre-initiation complex involves the action of the DNA-binding factor and promoter-selective factor, which recruits the TATA-binding protein (TBP) and other factors that activate RNA-pol I.

The role of eukaryotic RNA polymerase I is to promote

Transcription of the major rRNA transcripts, but not the 5S rRNA

Eukaryotic RNA processing: Alternative Splicing

allows multiple proteins to be made from a single gene regulated by a system of proteins (activators and repressors) that bind to "sequence elements" (enhancers and silencers) on the pre-mRNA transcript itself, which promote or reduce the usage of a particular splice site

Transcription factors (TFs) and transcriptional activators (regulators of chromatin)

are required to recruit RNA polymerase to DNA. (Unlike prokaryotes, where polymerase is bound to DNA and the Sigma factor recognizes the promoter)

Transcription factors (TFs)

bind to DNA sequences and regulate RNA polymerase

Transcription is often altered in

disease states

Writer

enzymes that add modifications to histone tails

Eraser

enzymes that remove the modifications of histone tails

Gene promoters are typically located

immediately upstream of the transcriptional start site (100-1000 base pairs)

Chromatin

important for transcription

Eukaryotic RNA processing: 5'-Capping of mRNA

initiates translation, prevents degradation 5' end of the RNA transcript contains a free triphosphate group since it was the first incorporated nucleotide in the chain, and this is replaced by a structure called a "cap" [cap binding complex] The last phosphate is removed on RNA by the enzyme RNA triphosphatase. The cap is then added by the enzyme Guanyl transferase, which catalyzes a reaction between the 5' end of the RNA transcript and a guanine triphosphate (GTP) molecule GTP is added in an inverted orientation so that the guanosine 5' end faces 5' end of the RNA chain (5' to 5' triphosphate linkage) Once in place, the cap plays a role in protein recognition of messenger RNA - important for further processing and translation

Poly A tail on 3' end

initiates translation, prevents degradation important for the nuclear export, translation, and stability of mRNA (protects the mRNA from degradation in the cytoplasm) Most polyadenylation sites start with the sequence AAUAAA (always poly A rich) The 3'-most segment of the newly made pre-mRNA is first cleaved off by a set of proteins; these proteins then synthesize the poly(A) tail at the RNA's 3' end. The poly(A) tail acts as the binding site for poly(A)- binding protein. Poly(A)-binding protein promotes export from the nucleus and translation and inhibits degradation.

Eukaryotic Promoters - RNA polymerase II transcriptional initiation

major enzyme that encodes mRNA Ø One of the roles for transcription Factors is to recruit a complex of proteins, called the Basal transcriptional complex, to initiate transcription. Ø The Basal transcriptional complex often recognizes the TATA box [TATA(A/T)(A/T)A], a relatively common promoter found at about 30 base pairs upstream of the transcription start site (about 10-25% genes) Ø TATA boxes are generally found in genes that exhibit noisy expression and is thought to act as an amplifier of expression Ø A protein called the TATA binding protein (TBP) is a general transcription factor that binds to the TATA box. (Note that TBP also binds to non-TATA box promoters as well)

Where does transcription occur in eukaryotes

nucleus

RNA editing

some cells can make discrete changes to specific nucleotide sequences within an RNA molecule after it has been generated by RNA polymerase Cytidine-to-Uridine (C-to-U) editing: (can help create a stop codon) Ø Cytidine deaminase - deaminates a cytidine base into a uridine base. Ø C-to-U editing is used for ApoB regulation (tissue specific expression of Cytidine deaminase) Adenosine-to-Inosine (A-to-I) editing: (I behaves like a G)

RNA polymerase II

synthesizes mRNA Ø Divided into a core region (often contains a TATA-box), defined as the minimal region capable of directing transcription in vitro, and a regulatory region. Ø The regulatory regions are highly varied in structure, reflecting the highly varied synthesis patterns of cellular proteins and the need for exquisite and complex regulation of these patterns.

Splicing

used to regulate mRNA regulated removal of introns of Pre-mRNAs concurrent with, or after, transcription. Splicing offers the opportunity to produce multiple mRNAs from one precursor, which can produce very different proteins (known as alternative splicing) mutations in splice sites are common in disease.

microRNAs (miRNAs)

they can be found anywhere in the genome and form hairpains that can turn into mature micro RNA eventally o Small non-coding single stranded RNA molecules ~21-25 nt o Unlike mRNA, vast majority of miRNAs are extremely stable: t1/2 ≈ 5 days function in the post-transcriptional regulation of gene function because each has a unique sequence 1) Transcribed by RNAPol II and part of a largeRNA precursors - precursor is called (pri-miRNA) 2) The Drosha complex processes pri-miRNA precursor hairpin to pre-miRNA *some pre-miRNAs are produced from very short introns (mirtrons) and can bypass the Drosha step. 3) Export to the cytoplasm 4) Additional processing by Dicer in the cytoplasm yields a mature ~22-bp double stranded miRNA. 5) Dicer initiates formation of RNA-induced silencing complex (RISC) containing the guide strand of the mature miRNA 6) miRNA exerts function after integration into active RISC complex by binding to complementary sites in mRNA targets. 7) Translational repression or Transcript degradation

RNA polymerase III

transcribes tRNA Ø Promoters are more varied in structure than the uniform RNA polymerase I promoters, and yet not as diverse as the RNA polymerase II promoters. Ø Some promoters rely on TATA-box, but many do not. have internal promoters within the transcribed region, which are recognized by the large complex that recruits TATA-binding protein (TBP) and the activating factors, which recruit and activate RNA-Pol III.

Other factors that help stimulate the Basal complex to increase transcription level

Ø Additional regions of DNA often recruit stimulatory factors help control the rate of transcription (e.g. proximal control elements) --o The CAAT box (GGCCAATCT consensus), that occur upstream by 60-100 bases to the initial transcription site --o G/C box (GGGCGG consensus) that occurs about 100-110 bases from the start site. Ø Genes that have the CAAT box often require the sequence for high levels of transcription

DNA modification

Ø DNA can be methylated on two bases: Cytosine and Adenine Ø DNA methylation of CG (also called CpG for 5'-C—phosphate—G-3' ) in promoters is generally associated with transcriptional repression Ø CpG can directly affect Transcription Factor binding and help recruit proteins that promote chromatin assembly - thereby making chromatin more compact (inactive)

Transcriptional Pausing

Ø RNA is transcribed at a heterogeneous rate (even from the same promoter) - this is the case in both Eukaryotes and Prokaryotes Ø Transcription is not continuous ⇒ interrupted by pausing events. Pausing can include halting transcription, or sometimes involves backtracking events. Ø During backtracking the catalytic site becomes disengaged from the 3′ end of RNA, rendering the RNA polymerase inactive, but stable. Ø Pausing plays important roles in coordinating translation and transcription in Prokaryotes. Ø Pausing plays a role in maintaining Transcriptional Fidelity (proofreading) Ø Pausing has key roles in overcoming roadblocks, such as nucleosomes Ø Pauses can fine tune transcription by mediating premature termination

Eukaryotic Transcriptional initiation: Promoters/Enhancers

Ø Regulatory elements known as enhancers (or silencers) are located several kilobases away (often > 100,000 base pairs) and control the activation/inhibition of promoters. Enhancer/silencers are located upstream, downstream or within the gene Ø Enhancers increase transcription rate, often facilitate DNA looping and function to make promoters accessible by directing the alteration of chromatin Ø Enhancer-binding TFs are called Activators, and these proteins often have two 2 conserved domains: i. DNA-binding domain at an enhancer ii. RNA polymerase-binding (or other protein factor-binding) domain that facilitates looping of DNA and chromatin remodeling

How splicing is regulated / reaction

Ø Splice donor: 5' end of intron: exon-G-U Ø Splice Acceptor: 3' end of intron: A-G-exon Ø Branch site: within the intron, about 30 nucleotides upstream of the splice acceptor, has an AT rich region with at least one A.

Basal transcriptional complex (RNA polymerase II)

Ø TFIID, a transcription factor, binds to TBP, followed by other general Transcription factors. Ø RNA polymerase can then recognize this multi-protein complex and bind to it, along with various other transcription factors, which together is known as the pre-initiation complex. Ø On of the components of the pre-initiation complex is a kinase (TFIIH), which phosphorylates RNA pol-II, triggering transcriptional activation Ø Transcription is then initiated, and the polymerase moves along the DNA strand

RNA polymerase recruitment: Transcription Factors

Ø TFs bind to specific DNA sequences Ø TFs usually respond to some signaling event that controls activity. Often there is a signal sensing domain (SSD) (e.g., a ligand binding domain), which senses external signals Ø Transcription factors are modular in structure and generally contain a DNA-binding domain (DBD) and a Transactivating domain (TAD), which contains binding sites for other proteins such as transcription co-regulators. Ø There are approximately 2600 proteins in the human genome that contain DNA-binding domains (DBDs), and most of these are presumed to function as transcription factors Ø Various combinations of TFs can provide context in biology

The spliceosome

Ø a large RNA-protein complex composed of five small nuclear ribonucleoproteins (snRNPs, pronounced 'snurps') Ø Assembly and activity of the spliceosome occurs during transcription of the pre-mRNA (IN THE NUCLEUS) Ø The RNA components of snRNPs interact with the intron and are involved in catalysis

Eukaryotic Transcription: Termination RNA polymerase II (mRNA)

• Polymerase II terminates transcription at random locations past the end of the gene being transcribed. Two protein complexes (FACTOR DEPENDENT) recognize a poly-A sequence, leading to cleavage of the RNA and resulting in a poly-A tail. • Template-independent addition of As at its new 3' end > polyadenylation > stabilizes RNA • Non-polyadenylated RNA transcripts are rapidly degraded

Eukaryotic Transcription: Termination RNA polymerase III (5S rRNA, tRNA, and other small RNAs)

• RNA Polymerase III terminates transcription in response to specific termination sequences in the newly synthesized RNA • RNA polymerase III can terminate transcription efficiently without involvement of additional factors (FACTOR INDEPENDENT). Often RNA that forms a hairpin is followed by a poly-U sequence (like the Rho-independent mechanism in prokaryotes)