Lecture 03/01P24 /

Prof Rod Devenish

RNA SYNTHESIS 2012

Transcription: DNADNA directed RNA Synthesis

RNA is transcribed from DNA Just one of the DNA strands for a gene is used to make the RNA; this strand is called the template strand. Different genes in the same DNA molecule need not use the same strand as the template. p RNA polymerase is the enzyme that uses DNA as a template to make RNA. p

Initiation of transcription
Initiation of transcription requires that RNA polymerase recognize, and bind po y e ase ecog e, a d b d tightly, a promoter sequence on DNA. g y Binding can be influenced by other proteins. p q The promoter sequence directs the RNA polymerase as to which of the two strands is the template and therefore in what direction the RNA polymerase should move.
See Figures Figure 14.7 Part 2 and 14.4 Part 1

Figure 14 4 DNA Is 14.4 Transcribed to Form RNA ( (Part 1) )

Figure 14 7 Transcription of 14.7 a Eukaryotic Gene (Part 2)

Figure 14 4 DNA Is 14.4 Transcribed to Form RNA ( (Part 2) )

RNA polymerase elongates the transcript

After binding, RNA polymerase unwinds binding the DNA about 10 base pairs at a time and reads the template (beginning at the initiation site) in the 3-to-5 direction. The RNA is synthesized (is elongated) in the 5 to 3 direction; i.e. the RNA transcript is antiparallel to the DNA template strand. See Figure 14.4 Part B

Figure 14 4 DNA Is 14.4 Transcribed to Form RNA ( (Part 3) )

RNA polymerase elongates the transcript

As the RNA transcript forms, it peels forms away, allowing the already transcribed DNA to be rewound into the double helix. Th energy f synthesis comes f The for th i from the removal and breakdown of the pyrophosphate group from each h h t f h nucleoside triphosphate building block added. (See Figure 11.21 Part a, Edn 7) dd d

Figure 11.21 Sequencing DNA

Transcription terminates at p particular base sequences q

P ti l Particular base sequences in the b i th

DNA specify termination. In eukaryotes, the process involves a protein complex and does not occur until a sequence signalling the site of eventual polyadenylation has been transcribed (See Figure 14.4 - Part C)

Figure 14 4 DNA Is 14.4 Transcribed to Form RNA ( (Part 3) )

The products of transcription

There are three major classes of RNA: M Messenger RNA (mRNA) - codes f a ( RNA) d for protein Ribosomal RNA (rRNA) - contributes to structure and function of ribosomes (protein synthesis machinery) ( ) y , Transfer RNA (tRNA) carry an amino, acid and act as an adaptor molecule allowing decoding of mRNA to p g g protein

RNA polymerases
Prokaryotes have one type of RNA polymerase that transcribes mRNA, mRNA tRNA, and rRNA Eukaryotes have three different RNA polymerases: I, II, and III. RNA polymerase II makes all mRNA in eukaryotes. RNA is not proof read and errors for proof-read RNA polymerases occur for every 104 to 105 bases incorporated. p

The Genetic Code

DNA codes for RNA by transcription. The genetic code consists of triplets of nucleotides (codons). Since there are four bases, there are 64 (4x4x4) possible codons. mRNA is read in codons. codons The 64 possible codons code for only 20 amino acids and the start and stop signals found in all mRNA molecules. See Figure 14 6 S Fi 14.6

Figure 14.6 The Genetic 14 6 Code

The Genetic Code

One mRNA codon, AUG (the start codon), indicates the starting p ) g point of translation and codes for methionine.
N.B. There is only one codon for methionine and AUG must therefore be used when a methionine occurs within a protein.

Three stop codons (UAA, UAG, and UGA) direct the ribosomes to stop reading the mRNA; that is, they indicate the end of translation. translation

The Genetic Code

After subtracting start and stop codons, th remaining 60 codons d the i i d code for 19 different amino acids. This means that many amino acids have more than one codon. codon Thus the code is redundant. See Figure 14 6 14.6.

The Genetic Code

The code is not ambiguous. Each codon is assigned only one amino acid, not acid two or three possible amino acids. Th genetic code i nearly universal, The ti d is l i l applying to all species on our planet. Minor variations are found within mitochondria and chloroplasts; other exceptions are few and slight.

Expression of the Eukaryotic Genome

E k Eukaryotic DNA (unlike prokaryotic ti ( lik k ti DNA), is separated from the cytoplasm by being contained within l b b i i d i hi a nucleus. The initial mRNA transcript of the (p ) DNA is modified (processed) before it is exported to the cytoplasm.
Review Figure 14.7 P R i Fi 14 7 Part 1

Figure 14 7 Transcription of 14.7 a Eukaryotic Gene (Part 1)

RNA Processing
After transcription, the pre-mRNA is altered by the addition of a methyl methylG cap at the 5 end and a poly A tail at the 3 end (following cleavage at 3 the AAUAAA sequence). These additions are important for the stability of the RNA and for its competence to be translated
Review Figure 14.10 g

Figure 14 10 Processing the 14.10 Ends of Eukaryotic Pre-mRNA Pre mRNA

RNA Processing
The introns are removed from the mRNA precursor by the spliceosome, a complex spliceosome of RNAs and proteins. A soon as the pre-mRNA is transcribed As th RNA i t ib d several small nuclear ribonucleoprotein particles (snRNPs) bind ti l ( RNP ) bi d This links all the coding regions together as a continuous sequence.
Review Figure 14 11 14.11

Figure 14 11 The 14.11 Spliceosome: An RNA Splicing Machine

Control of gene expression in eukaryotes

Eukaryotic gene expression can be controlled at the: transcriptional, transcriptional posttranscriptional, translational, and posttranslational levels.
Review Figure 16.13 16 13

Figure 16.13 Potential Points 16 13 for the Regulation of Gene Expression (Part 1) p ( )

Figure 16.13 Potential Points 16 13 for the Regulation of Gene Expression (Part 2) p ( )

Figure 16.13 Potential Points 16 13 for the Regulation of Gene Expression (Part 3) p ( )

Transcriptional Control
Th major method of control is selective The j th d f t li l ti transcription, which results from specific proteins (transcription factors) b d g to regulatory t a sc pt o acto s binding egu ato y regions on DNA. Specific transcription factors must bind to the promoter before RNA polymerase can bind. t b f l bi d A group of general transcription factors (regulatory proteins required for transcription) work with RNA polymerase II to transcribe mRNAs Initiation also depends on the proteins bound (activators and repressors).
Review Figures 16.14 and 16.15

Figure 16.14 The Initiation 16 14 of Transcription in Eukaryotes

Figure 16 15 Transcription 16.15 Factors, Repressors, Factors Repressors and Activators

Transcriptional Control
The simultaneous control of widely separated genes is id l t d i possible through proteins that bind to common sequences in their promoters.
Review Figure 16.17

Figure 16.17 Coordinating 16 17 Gene Expression

Post-transcriptional Post transcriptional Control

Some 50% of eukaryotic genes generally have several exons and ll h l d alternative splicing can be used to produce different proteins.
Review Figure 16.22

Figure 16.22 Alternative 16 22 Splicing Results in Different Mature mRNAs and Proteins

Post-transcriptional Post transcriptional Control

The stability of mRNA in the cytoplasm can be regulated by t l b l t db the binding of proteins. Specific AU-rich sequences mark some mRNAs for rapid breakdown by a ribonuclease complex (the exosome) )

RNAs involved in regulating g gene expression p

One class are microRNAs (miRNAs) ( ) - short, 19-25 nucleotide, nong coding RNAs Some miRNAs bind complementary sequences found in other mRNAs and prevent their translation and facilitate degradation. degradation
Review Figure 16.23 R i Fi 16 23

Figure 16 23 mRNA 16.23 Inhibition by MicroRNAs

miRNAs and regulation of g gene expression p

The precise number of miRNA genes in the human genome is still unknown; current estimates range from 500 g 1000 miRNAs may regulate 10 30% of human 10-30% genes; thus on average each miRNA might regulate about 200 target genes Many genes have target sites for a few different miRNAs

miRNAs in human disease

miRNAs appear to be de-regulated in in several human malignancies
including chronic lymphocytic leukemia, pediatric Burkitts lymphoma, gastric cancer and lung cancer

The miR-17-92 miRNA cluster on chromosome 13 is frequently amplified in B-cell lymphomas Elevated levels of these miRNAs are found in clinical samples and in related cell lines When these miRNAs are overexpressed in a mouse model cancer results

Profiling miRNA expression

Confirm that many miRNAs are tissuespecifically expressed p y p Potential for diagnostic use:
- comparing cancer and normal cells - distinguishing between related cancers

Translation of mRNA can be regulated

Translational control
Often there is little correlation between the amount of mRNA and the amount of protein synthesized. The concentrations of these proteins must be determined by factors acting after mRNA is made. made One way is binding of repressor proteins to the mRNA to inhibit translation. translation

Post-translational Post translational Control

Regulation of protein longevity:
Most gene products (proteins) are modified after translation. Regulating the lifetime of a protein is a way to control its actions. Proteins identified for breakdown are often linked to a 76-amino acid protein, ubiquitin. Other ubiquitin chains then attach to the primary one.

Post-translational Post translational Control

The proteinubiquitin complex then binds a complex called a proteasome proteasome. Inside the proteasome, energy from ATP is used to cut off the ubiquitin (for recycling) and unfold its targeted protein. Proteases di digest the protein into small h i i ll peptides and amino acids.
Review Figure 16 24 16.24

Figure 16 24 A Proteasome 16.24 Breaks Down Proteins