The number of base pairs (bp) in each nucleus in each of our more than 100 trillion somatic cells is 3 billion bp. Contained within each of our nuclei there is about 6 feet of highly coiled DNA. The DNA from each of our cells contains all the information necessary for the development of a new individual. However, each cell expresses certain genes specific to that cell that makes up a specific tissue (in the case of a multicellular organism). Thus, regulating eukaryotic gene expression is an extraordinarily important and complex event.

Regulation of gene expression in eukaryotic cells occurs at distinct levels:

• Genomic level (described in lecture 4)
• Thanscriptional level (the level of mRNA, rRNA or tRNA synthesis)
• RNA Processing level (the modification of mRNA, rRNA and tRNA)
• Translational level
• Post-translational level

Regulation at the transcriptional level

Transcription in eukaryotes is carried out by three distinct RNA Polymerases: RNA Pol I, RNA Pol II and RNA Pol III.

Transcriptional control is governed by the actions of a large number of proteins, called transcription factors (TF)

Promoters

Class II Promoters

• Class II promoters contain an initiator sequence, a TATA box, and upstream elements (GC box or a CCAAT box), and a downstream element. Many natural promoters lack at least one of these elements.
• The TATA box is the most common element and is about -25bp. In general, specialized genes that encode proteins made only in certain types of cells, have TATA box.
• TATA-less promoters exist for housekeeping genes and developmentally regulated genes such as the homeotic genes active during development.
• TATA box is involved in positioning the start of transcription. TATA Binding Protein (TBP) binds here.
• Upstream elements are GC boxes at about -47 to -61 and -80to -105. Sp1 binds here. The GC boxes are orientation-independent, but not position independent.

Class I Promoters

• RNA Polymerase I synthesizes the rRNA precursor gene. This gene is present in hundreds of copies in each cell, but each copy is virtually the same with the same promoter.
• The promoter has two critical regions, the core element, located -45 to +20 and the upstream control element (UCE) -156 to -107. The spacing between these two elements is crucial for proper transcription.

Class III Promoters

• Internal promoters occur for the 5S rRNA, tRNA or VA RNA genes. The internal promoter for rRNA consist of Box A (at +50), a short intermediate sequence and Box C (at +83). The internal promoter for tRNA and VA RNA genes has box A and box B.
• The 7SLRNA class III gene contains a weak internal promoter and a sequence in the 5' flanking region of the gene necessary for transcription.
• Other class III genes, 7SK and U6 RNA genes lack internal promoters and contain promoters that strongly resemble class II promoters. That is, these promoters exist in the 5' flanking region and contain TATA boxes.

General Transcription Factors

These factors attract the RNA polymerases (I,II and III) to their respective promoters, but only to a weak extent. Thus, they only stimulate a basal level of transcription.

Class II Factors

• Class II preinitiation complex consists of RNA Polymerase II, and six general transcription factors: TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH.
• First, TFIID binds to the TATA box with the aid of TFIIA. Second, TFIIB binds. Third, TFIIF helps RNA pol II bind to the DAB complex. Finally, TFIIE and TFIIH bind to the complex. TFIID contains the TATA box binding protein (TBP) and 8-10 TBP-associated factors (TAF'II's).
• TATA-less promoters can not bind TBP directly, but TAF'II's secure the whole TFIID binding to the promoter.

Class I Factors

• The preinitiation complex that forms at rRNA promoters involves polymerase I and two transcription factors: SL1 and Upstream binding factor (UBF).
• SL1 and UBF act synergistically to stimulate transcription.
• SL1 determines species specificity in rRNA gene transcription. SL1 consists of TBP and 3 TAFs. Thus, TBP functions with class I promoters, even though they lack TATA boxes.
• SL1 binds to the CORE site on the Class I promoters.
• UBF recognizes UCE site on the Class I promoters.

Class III Factors

• Transcription of all class III genes requires TFIIIB and TFIIIC. Transcription of the 5SrRNA genes also require TFIIIA.
• TFIIIC (and TFIIIA for 5SrRNA) binds to the internal promoter. These assembly factors then allow TFIIIB to bind to the upstream region. TFIIIB then helps polymerase III bind at the transcription start site. Finally, the polymerase transcribes the gene, probably removing TFIIIC (or A and C) in the process, but TFIIIB remains bound, so it can continue to promote further rounds of transcription.
• TFIIIB contains TBP and TAFs.

TBP is a universal transcription factor required by all three classes of genes.

Enhancers/Silencers

• In addition to the promoters, there are other regulatory sequences in the DNA, the enhancer. Enhancers are DNA sequences and are unique in that they can be moved experimentally from one place to another within the DNA molecule, or even inverted without affecting the ability of a bound transcription factor to stimulate transcription. Deletion of an enhancer can decrease the level of transcription by 100 fold or more.
• A transcription factor bound to an enhancer can stimulate transcription by one of the following ways: 1) Inducing changes in the chromatin thereby promoting the assembly of the basal transcription machinery at the promoter region. 2) Interacting with components of the basal transcription machinery.

Post-transcriptional gene regulation: RNA Processing

mRNA

RNA Polymerase II synthesizes the precursor to messenger RNA, the heterogeneous nuclear RNAs (hnRNAs) or the pre-mRNA.

Processing of mRNA:

• Once the 5' of a messenger RNA precursor has been synthesized, a methylated guanosine cap (7-methylguanosine cap) is added to this end. The functions of this cap are the following: 1) to prevent the 5' end of the mRNA from being digested by exonucleases,; 2) it aids in the transport of the mRNA out of the nucleus; 3) it plays an important role in the initiation of mRNA translation.
• Towards the 3' end, an endonuclease recognizes the sequence AAUAAA in the recently transcribed RNA, and cleaves the pre-mRNA about 15 nucleotides downstream from the recognition site. Following cleavage by the nuclease, an enzyme called poly(A) polymerase adds 250 or so adenosines to the 3' end. The poly(A) tail protects the mRNA from premature degradation by exonucleases.
• After capping and poly (A) addition, the pre-mRNA is spliced. The splicing of the pre-mRNA is done to remove introns. In order for RNA splicing to occur there need to be specific nucleotide sequences in the intron to be spliced out. The G/GU at the 5' end of the intron, the branch point "A" and the AG/G at the 3' end of the intron are present in the vast majority of eukaryotic pre-mRNAs. Processing occurs as each intron of the pre-mRNA becomes associated with a macromolecular complex called a spliceosome. Each spliceosome consists of a variety of proteins and a number of distinct ribonucleoproteins particles, called snRNPs (pronounced snurps) because they are composed of snRNAs bound to specific proteins. Many primary transcripts can be processed by two or more pathways, this is called alternate splicing, the same gene can code for more than one polypeptide.

rRNA

• The rRNAs synthesized by RNA Pol I are the 28S, 18S and 5.8S rRNAs. These are transcribed into a single primary transcript (the pre-rRNA). The 5S rRNA is synthesized from a separate RNA precursor outside the nucleolus.
• Before the pre-rRNA is cleaved into the 28S, 18S and 5.8S rRNAs, the pre-rRNA is heavily methylated and many of its uridines are modified to pseudouridines.
• The nucleolus is the site of rRNA processing and assembly of the two ribosomal subunits.
• The 5S rRNA, which is synthesized by RNA Pol III, is encoded by a large number of identical genes that are separate from the other rRNA genes and are located outside the nucleolus.

tRNA

• Transfer RNAs are synthesized by RNA Pol III and are synthesized from genes that are found in small clusters scattered around the genome. The primary transcript of a transfer RNA molecule is larger than the final product, and pieces on both, the 5' and 3', sides of the precursor tRNA must be "trimmed" away. In addition, extensive nucleotide modification occurs and the addition of CCA at the 3' end occurs.

Translational level control

The translation level control of gene expression depends on mRNA localization, mRNA translation, and mRNA stability.

Cytoplasmic localization of mRNA.

• The information that governs the cytoplasmic localization of a mRNA is located in the 3'untranslated region (UTR). Although the mechanism of mRNA localization is not well understood, it is thought to be mediated by proteins that recognize localization sequences in the mRNA. Microtubules and microfilaments have both been implicated in the accumulation of mRNAs in particular parts of a cell.

The control of mRNA translation.

• Translation can be controlled by 1) masking RNA so that it can not be translated; 2) translation can be affected by phosphorylating eIF2 and preventing translation of mRNA; 3) translation can be regulated by specific repressors that may bind to the 5' UTR or the mRNA.

Control of mRNA Stability.

• The longer a mRNA is present in a cell, the more times it can serve as a template for the assembly of a polypeptide. If a cell is to control gene expression, it is just a s important to regulate the survival of a mRNA as it is to regulate the synthesis of that mRNA in the first place. Unlike prokaryotic mRNA, which begin to be degraded at their 5' end even before their 3' end has been completed, most eukaryotic mRNAs are relatively long lived.
• It seems that the longevity of a mRNA is related to the length of its poly A tail. The poly A tail is not present as a naked RNA but is bound by a specific protein, the poly A binding protein (PABP). The poly A tail is not the only sequence responsible for the longevity of a mRNA molecule. The 3' UTR may contain either stabilizing sequences such as CCUCC repeats or destabilizing sequences such as AUUUA. These sequences are thought to bind proteins that either stabilize or destabilize the mRNA molecule.
• As a mRNA remains in the cytoplasm, its poly A tail tends to be gradually reduced in length as it is nibbled away by the poly A ribonuclease. No effect on the stability of the mRNA is observed until the tail becomes reduced to approximately 30 residues, which is probably too short a length to retain bound PABP molecules. For a mRNA to be degraded, a Poly(A) nuclease must first remove nucleotides from the 3' end. Once the 3' tail is removed, the message is decapped and degraded from the 5' end toward the 3' end.
• The half life of hnRNAs is of only a few minutes, whereas, the half life of rRNAs and tRNAs are measured in days or weeks. The half life of mRNAs range from about 15 min. to a period of days.