mRNA, tRNA and rRNA Synthesis

Post transcriptional modifications: ______ is not coded in the DNA ______ is coded in the DNA

poly A tail poly-adenilation signal

*Comparison of the steps leading from gene to protein in eukaryotes and bacteria.*

(A) In eukaryotic cells, the mRNA molecule resulting from transcription contains both coding (exon) and noncoding (intron) sequences. Before it can be translated into protein, the two ends of the RNA are modified, the introns are removed by an enzymatically catalyzed RNA splicing reaction, and the resulting mRNA is transported from the nucleus to the cytoplasm. For convenience, the steps in this figure are depicted as occurring one at a time; in reality, many occur concurrently. For example, the RNA cap is added and splicing begins before transcription has been completed. Because of the coupling between transcription and RNA processing, intact primary transcripts—the full-length RNAs that would, in theory, be produced if no processing had occurred—are found only rarely. (B) In prokaryotes, the production of mRNA is much simpler. The 5' end of an mRNA molecule is produced by the initiation of transcription, and the 3' end is produced by the termination of transcription. Since prokaryotic cells lack a nucleus, transcription and translation take place in a common compartment, and the translation of a bacterial mRNA often begins before its synthesis has been completed.

Trans Regulators of Transcription (Eukaryotes) *don't have to know the TF's*

*Transcription factors (TF)* (or basal factors) bind to TATA box and facilitate binding of RNA polymerase. -- TATA-binding protein (TBP) binds to TATA box. -- TF A & B bind TBP -- RNA polymerase binds, then factors E,F, & H bind. -- Can transcribe at a basal level, essential, cannot be regulated *Activator* (Transcriptional transactivators), and possibly repressors, communicate with basal factors through coactivators (linked in tight complex to TBP

*Know how tRNA is synthesized*

-- Eukaryotic cells contain multiple copies of each tRNA gene. -- RNA polymerase III transcribes the tRNA genes, recognizing a split promoter within the transcribed region of the gene. -- tRNA precursors are about 100 nucleotides -- Primary transcript assumes cloverleaf shape and is cleaved at 5' and 3' ends some contain introns that are spliced -- Bases are modified post-transcriptionally -- CCA sequence is added at 3'-end one at a time by nucleotidyltransferase. -- tRNAs exits nucleus

Structure of Ribosomal RNA and ribosomes

-- Ribosomes are subcellular compartment where protein synthesis occurs. -- Prokaryotic ribosomes contain three types of rRNA molecules -- The 50S and 30S subunits form the 70S ribosomes that participates in protein synthesis. -- Cytoplasmic ribosomes from eukaryotic cells contain 4 types of rRNA molecules. -- The 40S and 60S ribosomal subunits combine to form the 80S ribosome that participates in protein synthesis. -- Mitochondrial ribosomes have sedimentation coefficient of 55S, are smaller than cytoplasmic ribosomes, and are more similar to bacterial ribosomes than eukaryotic ribosomes. -- rRNAs in ribosomes exhibit extensive secondary structures.

tRNA Function

-- tRNA molecules carry amino acids to ribosomes and ensure that the amino acids are incorporated into the appropriate positions in the growing polypeptide chain; they are adaptors. -- Cells contain at least 20 different tRNA molecules, one for each amino acid -- Many amino acids have more than one tRNA.

tRNA Structure

-- tRNA molecules contain about 80 nucleotides. -- Each tRNA has one binding site for a specific sequence of three nucleotides in mRNA (the anticodon site) -- The sequence CCA, found at the 3' end, is the attachment site for the amino acid -- About 20% of the nucleotides are unusual and are produced by posttranscriptional modifications. -- The nucleosides dihydrouridine (D), ribothymidine (T) and pseudouridine are present in most tRNAs. -- Although their base sequences differ, tRNA molecules all form a similar cloverleaf structure.

Eukaryotes - Silencers

??????? She really didn't explain it

*Know how RNA is synthesized, including the reaction catalyzed by RNA polymerase and how it differs from DNA polymerase*

An overview of transcription at the site of RNA synthesis 1. Transcription begins with opening and unwinding of a small portion of the DNA double helix to expose the bases on each DNA strand. 2. One of the two strands of the DNA double helix then acts as a template for the synthesis of an RNA molecule. 3. The RNA strand does not remain hydrogen-bonded to the DNA template strand. 4. Instead, just behind the region where the ribonucleotides are being added, the RNA chain is displaced and the DNA helix re-forms.

*Know the general structure of a eukaryotic gene encoding for a protein, and how it differs from prokaryotic genes*

Comparison of structures of prokaryotic and eukyarotic mRNA The 5' and 3' ends of a bacterial mRNA are the unmodified ends of the chain synthesized by the RNA polymerase, which initiates and terminates transcription at those points, respectively. The corresponding ends of a eukaryotic mRNA are formed by added a 5' cap and by cleavage of the pre-mRNA transcript and the addition of a polyA tail, respectively. Bacterial mRNAs can contain the instructions for several different proteins whereas eukaryotic mRNAs nearly always contain the information for only a single protein.

THE FLOW OF GENETIC INFORMATION (central dogma)

Genes specify the kinds of proteins that are made by cells DNA is not the direct template for protein synthesis The templates for protein synthesis are RNA molecules Messenger RNA (mRNA) is the information carrying intermediate Transfer RNA (tRNA) and ribosomal RNA (rRNA) are part of the protein synthesizing machinery RNA synthesis (transcription) is carried out by RNA polymerase Transcription is regulated

Bacterial rRNA and tRNA transcripts

In prokaryotes, rRNA is produced as a single, long transcript that is cleaved to produce the 16S, 23S and 5S ribosomal RNAs. tRNA is also cleaved from a larger transcript. RNaseP, one of the cleavage enzymes, is a protein containing an RNA molecule. This RNA actually catalyzes the cleavage reaction.

The Fibrous Region???

In the nucleoulus... ???? The nucleolus consists of a fibrous region in which the genes for rRNA are being transcribed and a granular region in which the precursors of rRNA, complexed with proteins, are being modified and trimmed.

Eukaryotes - Enhancers

Other DNA elements (enhancers and silencers) regulate the frequency of transcription of genes; they are position independent Enhancers: --sequences are dissimilar --may be located thousands of base pairs from start point of transcription --orientation independent --position independent --*bind proteins (transcriptional tranactivators) which cause loops to form in DNA (thus brought into proximity with promoter)* --Transcriptional transactivators bound to enhancer seq stimulate transcription via coactivators

*Know how RNA polymerase differs from DNA polymerase*

RNA Polymerases can work de novo (without primer) --Bacterial cells have a single RNA polymerase that transcribes all types of RNA. --The RNA polymerase of Escherichia coli contains four subunits (2'), forming the core enzyme. --The sigma subunit confers specificity for binding. There are a number of different sigma factors that recognize the promoter regions of different groups of genes. --Eukaryotic cells have 3 RNA polymerases. --They all have same mechanism of action; however, they recognize different types of promoters.

Processing heterogeneous nuclear RNA (hnRNA)

RNA polymerase II binds sequences in the 5' flanking region and transcribes the gene, producing a primary transcript that is processed in the nucleus to produce the mature mRNA. Processing of the primary transcript involves --capping of the 5' end --addition of poly (A) tail to 3' end --methylation of some adenosine residues --removal of introns followed by splicing exons

Regulation of Transcription

RNA polymerase must determine: --when to transcribe a gene --where to initiate transcription --which strand of DNA to use as template --where to stop transcription --how fast to transcribe a gene

Splicing

Splicing: Eukaryotic transcripts contain introns and exons. Exons appear in the mature mRNA; introns are removed and exons are spliced together.

*Know how rRNA is synthesized*

Synthesis of Eukaryotic rRNA (I) : Ribosomal RNA is transcribed as a large precursor molecule that is then modified and trimmed to form the mature species that participates in protein synthesis. RNA polymerase I transcribes the 45S ribosomal RNA precursor which is cleaved to produce the 18S, 28S and 5.8S rRNAs. The promoter consists of two sequences in the 5'-flanking region of the gene that differ from the promoters for RNA polymerases II and III. The 45S precursor is methylated by S-adenosyl methionine (SAM) and cleaved to produce a 41S precursor, which undergoes further processing. (A small number of the nucleotides [1 to 2% of total] are methylated, usually on the 2'-hydroxyl of the ribose moieties.) During processing the rRNA precursors are associated with proteins.

Synthesis of Eukaryotic Ribosomes (II)

The genes for the 45S rRNA precursor are found in the nucleolar region of the nucleus. About 1,000 of these genes are present in the human genome and are linked in tandem. The 5S rRNA, produced by RNA polymerase III from genes located outside the nucleolus, migrates into the nucleolus and joins the ribonucleoprotein particles. The nucleolus consists of a fibrous region in which the genes for rRNA are being transcribed and a granular region in which the precursors of rRNA, complexed with proteins, are being modified and trimmed. Ultimately, the 40S and 60S ribosomal subunits are produced, which migrate through the nuclear pores to the cytoplasm where they combine, forming the 80S ribosomes which, complexed with mRNA, serve as the sites of protein synthesis

Prokaryote mRNA

The transcriptional unit is organized as an operon. Preceding the structural genes are regulatory regions of the operon known as the promoter and the operator.

Transcription

Transcription (synthesis of RNA from a DNA template) is catalyzed by RNA polymerase Eukaryotes --primary product of transcription is modified and trimmed --RNA must travel from nucleus to cytoplasm for translation --Three RNA polymerases: *Polymerase I - transcribes large rRNAs* *Polymerase II - transcribes mRNA* *Polymerase III -transcribes small RNAs (tRNA and 5S rRNA)* Prokaryotes --no nuclei, so transcription and translation may occur simultaneously --*have single RNA polymerase*

*Know how mRNA is synthesized* Prokaryotic

Transcription of Prokaryotic mRNA --The sigma factor is released when the transcript is about 10 nucleotides long. --Elongation continues until there is a transcription termination signal. --One type of signal is the formation of a hairpin loop in the transcript, preceding a number of U residues. --The second is the binding of a protein, the rho factor, which causes release of the RNA polymerase. --In many instances polycistronic mRNA is produced, and each cistron is translated into its cognate protein.

Eukaryotic promoter

Variety of consensus sequences: --In -25 region TATA box (12.5% of genes); --Other consensus sequences farther upstream or downstream after transcriptional start signal.

The Granular Region????

Where the cutting happens The nucleolus consists of a fibrous region in which the genes for rRNA are being transcribed and a granular region in which the precursors of rRNA, complexed with proteins, are being modified and trimmed.

Which is the coding strand for this sequence of RNA? 5'-AAUGCAUUGGCC-3' a. 5'-AATGCATTGGCC-3' b. 5'-TTACGTAACCGG-3' c. 5'-UUACGUAACCGG-3' d. 5'-GGCCAATGCATT-3'

a. 5'-AATGCATTGGCC-3'

The following sequences are found in the eukaryotic primary transcript: a. Polyadenylation sequence at terminus of most eukaryotic mRNAs b. Polyadenylation signal, AAUAAA c. Sequences for cap structure d. TATA box

b. Polyadenylation signal, AAUAAA

Rifampicin is an effective antibiotic because it: a. Inhibits ribosomal RNA synthesis b. Prevents initiation of bacterial RNA synthesis c. Intercalates within DNA d, Inhibits eukaryotic RNA polymerase II

b. Prevents initiation of bacterial RNA synthesis

RNA polymerase: a. Copies the DNA template in the 5' to 3' direction b. Synthesizes a single-stranded RNA molecule in 5' to 3' direction c. Requires a primer to synthesize a new RNA chain d. Can copy RNA to DNA

b. Synthesizes a single-stranded RNA molecule in 5' to 3' direction

Choose the correct statement: a. All eukaryotic ribosomal transcripts are synthesized in the nucleolar organizer b. The genes for the 45S rRNA precursor are linked in tandem in the human genome c. rRNA complexes with proteins only after exiting the nucleus d. There are no methylated bases in the RNA of eukaryotic ribosomes

b. The genes for the 45S rRNA precursor are linked in tandem in the human genome

Which sequence does eukaryotic DNA encode for? a. Polyadenylation sequence found at terminus of most eukaryotic mRNAs b. Sequences for cap structure c. TATA box

c. TATA box

Which is the template strand for this sequence of RNA? 5'-AAUGCAUUGGCC-3' a. 5'-AATGCATTGGCC-3' b. 5'-TTACGTAACCGG-3' c. 5'-UUACGUAACCGG-3' d. 5'-GGCCAATGCATT-3'

d. 5'-GGCCAATGCATT-3'

Choose the statement that is true for prokaryotic cells but not for eukaryotic cells: a. The cells have three RNA polymerases, each one transcribing a specific type of RNA. b. All RNA polymerases have the same mechanism of action. c. Transcription and translation cannot occur simultaneously. d. A single promoter can control the transcription of an operon containing multiple cistrons.

d. A single promoter can control the transcription of an operon containing multiple cistrons.

Inhibitors of RNA Synthesis

some bind to DNA --Actinomycin D intercalates DNA, blocks replication and transcription. Very toxic for both prokaryotic and eukaryotic cells. some bind to RNA polymerase -- rifampicin binds to bacterial RNA pol, prevents initiation of RNA synthesis, does not block elongation. Used as antibiotic. -- Streptolydigin binds to bacterial RNA polymerase and prevents elongation of RNA chains. Used as antibiotic. -- alpha-Amanitin inhibits eukaryotic RNA polymerase (Pol II readily inhibited, Pol I is insensitive). Found in poisonous mushrooms